miseq  (Illumina Inc)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Miseq Reagent kit V2
    Description:

    Catalog Number:
    MS102-2003
    Price:
    None
    Score:
    85
    Buy from Supplier


    Structured Review

    Illumina Inc miseq
    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina <t>MiSeq</t> sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent <t>PGM</t> sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    https://www.bioz.com/result/miseq/product/Illumina Inc
    Average 99 stars, based on 6838 article reviews
    Price from $9.99 to $1999.99
    miseq - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    2) Product Images from "Sequence Depth, Not PCR Replication, Improves Ecological Inference from Next Generation DNA Sequencing"

    Article Title: Sequence Depth, Not PCR Replication, Improves Ecological Inference from Next Generation DNA Sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090234

    Taxonomic assignment to OTUs observed between both sequencing platforms is highly consistent. Bar chart indicating the proportional richness and abundance of taxa identified to the class level in 55 soil samples sequenced with both 454 and Illumina MiSeq.
    Figure Legend Snippet: Taxonomic assignment to OTUs observed between both sequencing platforms is highly consistent. Bar chart indicating the proportional richness and abundance of taxa identified to the class level in 55 soil samples sequenced with both 454 and Illumina MiSeq.

    Techniques Used: Sequencing

    Taxon abundance is strongly correlated across sequencing runs with different levels of PCR replication. Circles represent individual taxa, and the relationship between the log 10 of their abundance in samples comprised of 16 PCR replicates (y axis) and one PCR replicate (x axis) for CT2 (top two panels), OR1 (middle two panels), and OR4 (bottom two panels). Dashed lines represent a 1∶1 relationship. The left three panels show samples sequenced with 454 and the right three panels show samples sequenced with Illumina MiSeq.
    Figure Legend Snippet: Taxon abundance is strongly correlated across sequencing runs with different levels of PCR replication. Circles represent individual taxa, and the relationship between the log 10 of their abundance in samples comprised of 16 PCR replicates (y axis) and one PCR replicate (x axis) for CT2 (top two panels), OR1 (middle two panels), and OR4 (bottom two panels). Dashed lines represent a 1∶1 relationship. The left three panels show samples sequenced with 454 and the right three panels show samples sequenced with Illumina MiSeq.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Patterns of α- and β-diversity are highly reproducible when samples are sequenced on different platforms. Regressions of diversity found in 55 soil samples sequenced on both platforms. Left three columns: points represent individual samples, and the relationship between the total richness per sample found when sequenced with 454 (y axis) and Illumina MiSeq (x axis). Right three columns: points represent pairwise differences in between-sample community composition and the relationship between dissimilarity found with 454 (y axis) and Illumina (x axis). Dashed lines represent the linear models predicting the relationships.
    Figure Legend Snippet: Patterns of α- and β-diversity are highly reproducible when samples are sequenced on different platforms. Regressions of diversity found in 55 soil samples sequenced on both platforms. Left three columns: points represent individual samples, and the relationship between the total richness per sample found when sequenced with 454 (y axis) and Illumina MiSeq (x axis). Right three columns: points represent pairwise differences in between-sample community composition and the relationship between dissimilarity found with 454 (y axis) and Illumina (x axis). Dashed lines represent the linear models predicting the relationships.

    Techniques Used:

    Estimated species richness does not depend on the number of PCR reactions pooled prior to sequencing. Plots of independent replicates representing different levels of PCR pooling for samples CT2, OR1, and OR4 against four different diversity indicators. Points are colored by sample ID. Dotted lines represent the average between different replicates of the same sample. The top three lines represent samples sequenced with Illumina MiSeq and the bottoms three lines represent the same samples sequenced with 454.
    Figure Legend Snippet: Estimated species richness does not depend on the number of PCR reactions pooled prior to sequencing. Plots of independent replicates representing different levels of PCR pooling for samples CT2, OR1, and OR4 against four different diversity indicators. Points are colored by sample ID. Dotted lines represent the average between different replicates of the same sample. The top three lines represent samples sequenced with Illumina MiSeq and the bottoms three lines represent the same samples sequenced with 454.

    Techniques Used: Polymerase Chain Reaction, Sequencing

    Broad ecological patterns of β-diversity are recovered equally well with each sequencing platform. Non-metric multidimensional scaling of fungal communities from 55 soil samples sequenced with both 454 (circles) and Illumina MiSeq (triangles). Points are colored by the three regions of sample collection. Ordinations are based on between-sample dissimilarity calculated with Jaccard (top panel), Bray-Curtis (middle panel), and β-sim (bottom panel).
    Figure Legend Snippet: Broad ecological patterns of β-diversity are recovered equally well with each sequencing platform. Non-metric multidimensional scaling of fungal communities from 55 soil samples sequenced with both 454 (circles) and Illumina MiSeq (triangles). Points are colored by the three regions of sample collection. Ordinations are based on between-sample dissimilarity calculated with Jaccard (top panel), Bray-Curtis (middle panel), and β-sim (bottom panel).

    Techniques Used: Sequencing

    Increasing sequence depth reduces pseudo-β-diversity. Plots of the between-sample Bray-Curtis dissimilarity (top two panels) and Jaccard dissimilarity (bottom two panels) in CT2, OR1, and OR4 against per-sample sequencing depth. Points represent the β-diversity values between different replicates of the same sample and are colored by sample ID. Dashed lines connect each symbol within a sample. The left two panels show samples sequenced with 454 and the right two panels show samples sequenced with Illumina MiSeq.
    Figure Legend Snippet: Increasing sequence depth reduces pseudo-β-diversity. Plots of the between-sample Bray-Curtis dissimilarity (top two panels) and Jaccard dissimilarity (bottom two panels) in CT2, OR1, and OR4 against per-sample sequencing depth. Points represent the β-diversity values between different replicates of the same sample and are colored by sample ID. Dashed lines connect each symbol within a sample. The left two panels show samples sequenced with 454 and the right two panels show samples sequenced with Illumina MiSeq.

    Techniques Used: Sequencing

    3) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    4) Product Images from "Evaluation of metagenetic community analysis of planktonic copepods using Illumina MiSeq: Comparisons with morphological classification and metagenetic analysis using Roche 454"

    Article Title: Evaluation of metagenetic community analysis of planktonic copepods using Illumina MiSeq: Comparisons with morphological classification and metagenetic analysis using Roche 454

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181452

    Total species and MOTU numbers of copepods in field-collected samples. MOTU numbers were calculated at 97 and 99% similarity thresholds in metagenetic analyses using both Illumina MiSeq and Roche 454 data.
    Figure Legend Snippet: Total species and MOTU numbers of copepods in field-collected samples. MOTU numbers were calculated at 97 and 99% similarity thresholds in metagenetic analyses using both Illumina MiSeq and Roche 454 data.

    Techniques Used:

    Order-level taxonomic compositions of copepods in field-collected samples between (A) species and MOTUs, and (B) biomass and sequence reads. Metagenetic analyses using both Illumina MiSeq and Roche 454 data were performed at a 97% similarity threshold.
    Figure Legend Snippet: Order-level taxonomic compositions of copepods in field-collected samples between (A) species and MOTUs, and (B) biomass and sequence reads. Metagenetic analyses using both Illumina MiSeq and Roche 454 data were performed at a 97% similarity threshold.

    Techniques Used: Sequencing

    Family-level taxonomic compositions of calanoid copepods in field-collected samples between (A) species and MOTUs, and (B) biomass and sequence reads. Metagenetic analyses using both Illumina MiSeq and Roche 454 data were performed at a 97% similarity threshold.
    Figure Legend Snippet: Family-level taxonomic compositions of calanoid copepods in field-collected samples between (A) species and MOTUs, and (B) biomass and sequence reads. Metagenetic analyses using both Illumina MiSeq and Roche 454 data were performed at a 97% similarity threshold.

    Techniques Used: Sequencing

    MOTU numbers at different minimum abundance thresholds of sequence reads: Comparisons of mock community analysis using (A) Illumina MiSeq and (B) Roche 454, and analyses of field-collected samples using (C) Illumina MiSeq and (D) Roche 454. A 0.05% of total reads for a single sample is indicated by dashed lines. Effects of the abundance threshold on MOTU numbers were evaluated both at a 97 and 99% similarity threshold for MOTU clustering.
    Figure Legend Snippet: MOTU numbers at different minimum abundance thresholds of sequence reads: Comparisons of mock community analysis using (A) Illumina MiSeq and (B) Roche 454, and analyses of field-collected samples using (C) Illumina MiSeq and (D) Roche 454. A 0.05% of total reads for a single sample is indicated by dashed lines. Effects of the abundance threshold on MOTU numbers were evaluated both at a 97 and 99% similarity threshold for MOTU clustering.

    Techniques Used: Sequencing

    5) Product Images from "A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses"

    Article Title: A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

    Journal: mSystems

    doi: 10.1128/mSystems.00039-15

    The ability of the SOP to sequence and identify unknown samples. (A) High-titer viral stocks were subjected to the SOP, anonymized, and shipped to JCVI for sequencing and data analysis. Samples were pooled and sequenced by Illumina MiSeq. Data from each corresponding sample were put into de novo assembly, and large contigs ( > 500 bp) were used to identify the best full-length viral genome references by nucleotide BLAST search against the NT database. Raw data were then mapped onto the best available reference genome. (B) Mapping coverage of an unknown sample against the selected genome for St. Louis encephalitis virus (SLEV). (C) Mapping coverage of an unknown sample against the selected genome for Western equine encephalitis virus (WEEV). (D) Mapping coverage of an unknown against the selected genome for Chikungunya virus (CHIKV). In panels B to D, nucleotide coverage depth is indicated on the y axis, and genomic position, with the length of each genome indicated as well as the best available reference genome, is indicated on the x axis.
    Figure Legend Snippet: The ability of the SOP to sequence and identify unknown samples. (A) High-titer viral stocks were subjected to the SOP, anonymized, and shipped to JCVI for sequencing and data analysis. Samples were pooled and sequenced by Illumina MiSeq. Data from each corresponding sample were put into de novo assembly, and large contigs ( > 500 bp) were used to identify the best full-length viral genome references by nucleotide BLAST search against the NT database. Raw data were then mapped onto the best available reference genome. (B) Mapping coverage of an unknown sample against the selected genome for St. Louis encephalitis virus (SLEV). (C) Mapping coverage of an unknown sample against the selected genome for Western equine encephalitis virus (WEEV). (D) Mapping coverage of an unknown against the selected genome for Chikungunya virus (CHIKV). In panels B to D, nucleotide coverage depth is indicated on the y axis, and genomic position, with the length of each genome indicated as well as the best available reference genome, is indicated on the x axis.

    Techniques Used: Sequencing, Western Blot

    The SOP generates high-quality full-genome sequence data across multiple ssRNA+ virus families. Pooled samples from the SOP were sequenced on the Illumina MiSeq platform. Samples were demultiplexed, the adaptors were trimmed, and low-quality sequencing reads were removed. Sequencing reads were mapped corresponding to input viruses. These viruses include foot-and-mouth disease virus (FMDV) type O (GenBank accession no. KF112887.1 ) (A), West Nile virus (WNV) AF404756.1 ) (B), human rhinovirus 16 (HRV-16) (GenBank accession no. L24917.1 ) (C), Chikungunya virus (CHIKV) (pJM6-3-CHIKV 181/25-mkate) (D), and Middle East respiratory syndrome coronavirus (MERS) (GenBank accession no. KJ614529.1 ) (E). Nucleotide coverage depth (NT coverage) is indicated on the y axis, and nucleotide (NT) position is indicated on the x axis. The genome length for each virus is indicated on the x axis, and the percentage of the genome covered greater than 3 nucleotides is indicated. For FMDV, WNV, HRV-16, and CHIKV, data represent material from a single barcode. For MERS, the data shown is a combination of four barcodes generated from the same sample.
    Figure Legend Snippet: The SOP generates high-quality full-genome sequence data across multiple ssRNA+ virus families. Pooled samples from the SOP were sequenced on the Illumina MiSeq platform. Samples were demultiplexed, the adaptors were trimmed, and low-quality sequencing reads were removed. Sequencing reads were mapped corresponding to input viruses. These viruses include foot-and-mouth disease virus (FMDV) type O (GenBank accession no. KF112887.1 ) (A), West Nile virus (WNV) AF404756.1 ) (B), human rhinovirus 16 (HRV-16) (GenBank accession no. L24917.1 ) (C), Chikungunya virus (CHIKV) (pJM6-3-CHIKV 181/25-mkate) (D), and Middle East respiratory syndrome coronavirus (MERS) (GenBank accession no. KJ614529.1 ) (E). Nucleotide coverage depth (NT coverage) is indicated on the y axis, and nucleotide (NT) position is indicated on the x axis. The genome length for each virus is indicated on the x axis, and the percentage of the genome covered greater than 3 nucleotides is indicated. For FMDV, WNV, HRV-16, and CHIKV, data represent material from a single barcode. For MERS, the data shown is a combination of four barcodes generated from the same sample.

    Techniques Used: Sequencing, Generated

    The SOP can detect WNV infection in vitro and in vivo . (A and B) Infected WNV cells were spiked into uninfected cells (A) or uninfected tissues (B), libraries were prepared on RNA according to the SOP, and the libraries were examined by Illumina MiSeq. (C and D) Footpad (C) and brain tissue (D) from WNV-infected mice were analyzed at 5, 10, and 29 days postinfection for WNV-specific sequence reads by Illumina HiSeq. (A) Data representing the ability of the SOP to identify WNV-specific reads from limiting dilutions of WNV-infected Vero cells spiked into uninfected 293T cells. Mapped and unmapped reads from each sample are displayed. (B) The SOP identifies WNV-specific reads from limiting dilutions of WNV-infected Vero cells spiked into uninfected mouse tissues (spleen and brain). Mapped and unmapped reads from each sample are shown. (C) WNV was detected in the footpad RNA of mice prepared according to the SOP at the indicated times postinfection. (D) WNV-specific reads can be detected from brain tissue RNA of mice at the indicated times postinfection. For panels C and D, three mice per group were analyzed, and WNV-mapped and unmapped reads are shown.
    Figure Legend Snippet: The SOP can detect WNV infection in vitro and in vivo . (A and B) Infected WNV cells were spiked into uninfected cells (A) or uninfected tissues (B), libraries were prepared on RNA according to the SOP, and the libraries were examined by Illumina MiSeq. (C and D) Footpad (C) and brain tissue (D) from WNV-infected mice were analyzed at 5, 10, and 29 days postinfection for WNV-specific sequence reads by Illumina HiSeq. (A) Data representing the ability of the SOP to identify WNV-specific reads from limiting dilutions of WNV-infected Vero cells spiked into uninfected 293T cells. Mapped and unmapped reads from each sample are displayed. (B) The SOP identifies WNV-specific reads from limiting dilutions of WNV-infected Vero cells spiked into uninfected mouse tissues (spleen and brain). Mapped and unmapped reads from each sample are shown. (C) WNV was detected in the footpad RNA of mice prepared according to the SOP at the indicated times postinfection. (D) WNV-specific reads can be detected from brain tissue RNA of mice at the indicated times postinfection. For panels C and D, three mice per group were analyzed, and WNV-mapped and unmapped reads are shown.

    Techniques Used: Infection, In Vitro, In Vivo, Mouse Assay, Sequencing

    Defining the sensitivity of the SOP on Illumina MiSeq and HiSeq platforms. RNA from serial 10-fold dilutions of an HRV-16 virion stock was treated according to the SOP. Samples were pooled and sequenced on an Illumina MiSeq or HiSeq platform. The left y axis denotes the number of reads mapped to the HRV-16 reference genome, the right y axis denotes the percentage of the reference genome covered, and the x axis denotes the input PFU for each reaction. The solid black line demonstrates that sequencing reads were detected between 1 and 10 PFU on the MiSeq platform. A similar sensitivity is obtained on the HiSeq platform, as denoted by the solid red line. The corresponding percentage of the HRV-16 genomic coverage from each platform is denoted by a dashed black line (MiSeq) and a dashed red line (HiSeq). The slight enhancement of genomic coverage on the MiSeq platform, despite the fewer number of sequence reads, results from the longer read length on the MiSeq platform (300 nucleotides [nt]) over the HiSeq platform (100 nt), as sequencing capacity is in excess at all dilutions.
    Figure Legend Snippet: Defining the sensitivity of the SOP on Illumina MiSeq and HiSeq platforms. RNA from serial 10-fold dilutions of an HRV-16 virion stock was treated according to the SOP. Samples were pooled and sequenced on an Illumina MiSeq or HiSeq platform. The left y axis denotes the number of reads mapped to the HRV-16 reference genome, the right y axis denotes the percentage of the reference genome covered, and the x axis denotes the input PFU for each reaction. The solid black line demonstrates that sequencing reads were detected between 1 and 10 PFU on the MiSeq platform. A similar sensitivity is obtained on the HiSeq platform, as denoted by the solid red line. The corresponding percentage of the HRV-16 genomic coverage from each platform is denoted by a dashed black line (MiSeq) and a dashed red line (HiSeq). The slight enhancement of genomic coverage on the MiSeq platform, despite the fewer number of sequence reads, results from the longer read length on the MiSeq platform (300 nucleotides [nt]) over the HiSeq platform (100 nt), as sequencing capacity is in excess at all dilutions.

    Techniques Used: Sequencing

    NGS on SOP-generated HRV-16-specific sequence from pure and mixed samples is slightly less sensitive than quantitative real-time RT-PCR (qrRT-PCR). Four independent tests were conducted to determine the sensitivity of the SOP. Test 1 detects HRV-16 sequence from dilutions of purified virus. Test 2 detects HRV-16 sequence from dilutions of genomic RNA. Test 3 detects HRV-16 sequence from dilutions of virus spiked into H1 HeLa cells. Test 4 detects HRV-16 sequence from genomic RNA dilutions spiked into total HeLa cell RNA. A ribosomal removal step was performed for tests 3 and 4 prior to the initiation of the SOP. For each sample, a fraction of the RNA used to initiate the SOP was subjected to qrRT-PCR analysis. (A to D) HRV-16-specific reads obtained by MiSeq (black solid lines) are plotted on the left y axis and the cycle threshold (Ct) values are plotted on the right y axis (red lines). Sequencing reads not mapping to the HRV-16 reference are also indicated (black dashed lines). Corresponding HRV-16 input PFU values are plotted on the x axis. (A) The limit of detection (LOD) for test 1 in this experiment is between 10 1 and 10 2 input PFU. The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU). (B) The LOD for test 2 in this experiment is between 10 1 and 10 2 input PFU. The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU). (C) The LOD for test 3 in this experiment is between 10 2 and 10 3 input PFU. The corresponding LOD by qrRT-PCR is approximately 100-fold greater (10 0 to 10 1 input PFU). (D) The LOD for test 4 in this experiment is between 10 1 and 10 2 input PFU; however, single reads are detected down to an input of 10 −1 . The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU) when individual HRV-16 reads are not considered and approximately 10-fold less sensitive when individual HRV-16 reads are considered.
    Figure Legend Snippet: NGS on SOP-generated HRV-16-specific sequence from pure and mixed samples is slightly less sensitive than quantitative real-time RT-PCR (qrRT-PCR). Four independent tests were conducted to determine the sensitivity of the SOP. Test 1 detects HRV-16 sequence from dilutions of purified virus. Test 2 detects HRV-16 sequence from dilutions of genomic RNA. Test 3 detects HRV-16 sequence from dilutions of virus spiked into H1 HeLa cells. Test 4 detects HRV-16 sequence from genomic RNA dilutions spiked into total HeLa cell RNA. A ribosomal removal step was performed for tests 3 and 4 prior to the initiation of the SOP. For each sample, a fraction of the RNA used to initiate the SOP was subjected to qrRT-PCR analysis. (A to D) HRV-16-specific reads obtained by MiSeq (black solid lines) are plotted on the left y axis and the cycle threshold (Ct) values are plotted on the right y axis (red lines). Sequencing reads not mapping to the HRV-16 reference are also indicated (black dashed lines). Corresponding HRV-16 input PFU values are plotted on the x axis. (A) The limit of detection (LOD) for test 1 in this experiment is between 10 1 and 10 2 input PFU. The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU). (B) The LOD for test 2 in this experiment is between 10 1 and 10 2 input PFU. The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU). (C) The LOD for test 3 in this experiment is between 10 2 and 10 3 input PFU. The corresponding LOD by qrRT-PCR is approximately 100-fold greater (10 0 to 10 1 input PFU). (D) The LOD for test 4 in this experiment is between 10 1 and 10 2 input PFU; however, single reads are detected down to an input of 10 −1 . The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU) when individual HRV-16 reads are not considered and approximately 10-fold less sensitive when individual HRV-16 reads are considered.

    Techniques Used: Next-Generation Sequencing, Generated, Sequencing, Quantitative RT-PCR, Polymerase Chain Reaction, Purification

    6) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    7) Product Images from "Sequence Depth, Not PCR Replication, Improves Ecological Inference from Next Generation DNA Sequencing"

    Article Title: Sequence Depth, Not PCR Replication, Improves Ecological Inference from Next Generation DNA Sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090234

    Taxonomic assignment to OTUs observed between both sequencing platforms is highly consistent. Bar chart indicating the proportional richness and abundance of taxa identified to the class level in 55 soil samples sequenced with both 454 and Illumina MiSeq.
    Figure Legend Snippet: Taxonomic assignment to OTUs observed between both sequencing platforms is highly consistent. Bar chart indicating the proportional richness and abundance of taxa identified to the class level in 55 soil samples sequenced with both 454 and Illumina MiSeq.

    Techniques Used: Sequencing

    Taxon abundance is strongly correlated across sequencing runs with different levels of PCR replication. Circles represent individual taxa, and the relationship between the log 10 of their abundance in samples comprised of 16 PCR replicates (y axis) and one PCR replicate (x axis) for CT2 (top two panels), OR1 (middle two panels), and OR4 (bottom two panels). Dashed lines represent a 1∶1 relationship. The left three panels show samples sequenced with 454 and the right three panels show samples sequenced with Illumina MiSeq.
    Figure Legend Snippet: Taxon abundance is strongly correlated across sequencing runs with different levels of PCR replication. Circles represent individual taxa, and the relationship between the log 10 of their abundance in samples comprised of 16 PCR replicates (y axis) and one PCR replicate (x axis) for CT2 (top two panels), OR1 (middle two panels), and OR4 (bottom two panels). Dashed lines represent a 1∶1 relationship. The left three panels show samples sequenced with 454 and the right three panels show samples sequenced with Illumina MiSeq.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Patterns of α- and β-diversity are highly reproducible when samples are sequenced on different platforms. Regressions of diversity found in 55 soil samples sequenced on both platforms. Left three columns: points represent individual samples, and the relationship between the total richness per sample found when sequenced with 454 (y axis) and Illumina MiSeq (x axis). Right three columns: points represent pairwise differences in between-sample community composition and the relationship between dissimilarity found with 454 (y axis) and Illumina (x axis). Dashed lines represent the linear models predicting the relationships.
    Figure Legend Snippet: Patterns of α- and β-diversity are highly reproducible when samples are sequenced on different platforms. Regressions of diversity found in 55 soil samples sequenced on both platforms. Left three columns: points represent individual samples, and the relationship between the total richness per sample found when sequenced with 454 (y axis) and Illumina MiSeq (x axis). Right three columns: points represent pairwise differences in between-sample community composition and the relationship between dissimilarity found with 454 (y axis) and Illumina (x axis). Dashed lines represent the linear models predicting the relationships.

    Techniques Used:

    Estimated species richness does not depend on the number of PCR reactions pooled prior to sequencing. Plots of independent replicates representing different levels of PCR pooling for samples CT2, OR1, and OR4 against four different diversity indicators. Points are colored by sample ID. Dotted lines represent the average between different replicates of the same sample. The top three lines represent samples sequenced with Illumina MiSeq and the bottoms three lines represent the same samples sequenced with 454.
    Figure Legend Snippet: Estimated species richness does not depend on the number of PCR reactions pooled prior to sequencing. Plots of independent replicates representing different levels of PCR pooling for samples CT2, OR1, and OR4 against four different diversity indicators. Points are colored by sample ID. Dotted lines represent the average between different replicates of the same sample. The top three lines represent samples sequenced with Illumina MiSeq and the bottoms three lines represent the same samples sequenced with 454.

    Techniques Used: Polymerase Chain Reaction, Sequencing

    Broad ecological patterns of β-diversity are recovered equally well with each sequencing platform. Non-metric multidimensional scaling of fungal communities from 55 soil samples sequenced with both 454 (circles) and Illumina MiSeq (triangles). Points are colored by the three regions of sample collection. Ordinations are based on between-sample dissimilarity calculated with Jaccard (top panel), Bray-Curtis (middle panel), and β-sim (bottom panel).
    Figure Legend Snippet: Broad ecological patterns of β-diversity are recovered equally well with each sequencing platform. Non-metric multidimensional scaling of fungal communities from 55 soil samples sequenced with both 454 (circles) and Illumina MiSeq (triangles). Points are colored by the three regions of sample collection. Ordinations are based on between-sample dissimilarity calculated with Jaccard (top panel), Bray-Curtis (middle panel), and β-sim (bottom panel).

    Techniques Used: Sequencing

    Increasing sequence depth reduces pseudo-β-diversity. Plots of the between-sample Bray-Curtis dissimilarity (top two panels) and Jaccard dissimilarity (bottom two panels) in CT2, OR1, and OR4 against per-sample sequencing depth. Points represent the β-diversity values between different replicates of the same sample and are colored by sample ID. Dashed lines connect each symbol within a sample. The left two panels show samples sequenced with 454 and the right two panels show samples sequenced with Illumina MiSeq.
    Figure Legend Snippet: Increasing sequence depth reduces pseudo-β-diversity. Plots of the between-sample Bray-Curtis dissimilarity (top two panels) and Jaccard dissimilarity (bottom two panels) in CT2, OR1, and OR4 against per-sample sequencing depth. Points represent the β-diversity values between different replicates of the same sample and are colored by sample ID. Dashed lines connect each symbol within a sample. The left two panels show samples sequenced with 454 and the right two panels show samples sequenced with Illumina MiSeq.

    Techniques Used: Sequencing

    8) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    9) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    10) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    11) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    12) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    13) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    14) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    15) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    16) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    17) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    18) Product Images from "A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses"

    Article Title: A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

    Journal: mSystems

    doi: 10.1128/mSystems.00039-15

    The ability of the SOP to sequence and identify unknown samples. (A) High-titer viral stocks were subjected to the SOP, anonymized, and shipped to JCVI for sequencing and data analysis. Samples were pooled and sequenced by Illumina MiSeq. Data from each corresponding sample were put into de novo assembly, and large contigs ( > 500 bp) were used to identify the best full-length viral genome references by nucleotide BLAST search against the NT database. Raw data were then mapped onto the best available reference genome. (B) Mapping coverage of an unknown sample against the selected genome for St. Louis encephalitis virus (SLEV). (C) Mapping coverage of an unknown sample against the selected genome for Western equine encephalitis virus (WEEV). (D) Mapping coverage of an unknown against the selected genome for Chikungunya virus (CHIKV). In panels B to D, nucleotide coverage depth is indicated on the y axis, and genomic position, with the length of each genome indicated as well as the best available reference genome, is indicated on the x axis.
    Figure Legend Snippet: The ability of the SOP to sequence and identify unknown samples. (A) High-titer viral stocks were subjected to the SOP, anonymized, and shipped to JCVI for sequencing and data analysis. Samples were pooled and sequenced by Illumina MiSeq. Data from each corresponding sample were put into de novo assembly, and large contigs ( > 500 bp) were used to identify the best full-length viral genome references by nucleotide BLAST search against the NT database. Raw data were then mapped onto the best available reference genome. (B) Mapping coverage of an unknown sample against the selected genome for St. Louis encephalitis virus (SLEV). (C) Mapping coverage of an unknown sample against the selected genome for Western equine encephalitis virus (WEEV). (D) Mapping coverage of an unknown against the selected genome for Chikungunya virus (CHIKV). In panels B to D, nucleotide coverage depth is indicated on the y axis, and genomic position, with the length of each genome indicated as well as the best available reference genome, is indicated on the x axis.

    Techniques Used: Sequencing, Western Blot

    The SOP generates high-quality full-genome sequence data across multiple ssRNA+ virus families. Pooled samples from the SOP were sequenced on the Illumina MiSeq platform. Samples were demultiplexed, the adaptors were trimmed, and low-quality sequencing reads were removed. Sequencing reads were mapped corresponding to input viruses. These viruses include foot-and-mouth disease virus (FMDV) type O (GenBank accession no. KF112887.1 ) (A), West Nile virus (WNV) AF404756.1 ) (B), human rhinovirus 16 (HRV-16) (GenBank accession no. L24917.1 ) (C), Chikungunya virus (CHIKV) (pJM6-3-CHIKV 181/25-mkate) (D), and Middle East respiratory syndrome coronavirus (MERS) (GenBank accession no. KJ614529.1 ) (E). Nucleotide coverage depth (NT coverage) is indicated on the y axis, and nucleotide (NT) position is indicated on the x axis. The genome length for each virus is indicated on the x axis, and the percentage of the genome covered greater than 3 nucleotides is indicated. For FMDV, WNV, HRV-16, and CHIKV, data represent material from a single barcode. For MERS, the data shown is a combination of four barcodes generated from the same sample.
    Figure Legend Snippet: The SOP generates high-quality full-genome sequence data across multiple ssRNA+ virus families. Pooled samples from the SOP were sequenced on the Illumina MiSeq platform. Samples were demultiplexed, the adaptors were trimmed, and low-quality sequencing reads were removed. Sequencing reads were mapped corresponding to input viruses. These viruses include foot-and-mouth disease virus (FMDV) type O (GenBank accession no. KF112887.1 ) (A), West Nile virus (WNV) AF404756.1 ) (B), human rhinovirus 16 (HRV-16) (GenBank accession no. L24917.1 ) (C), Chikungunya virus (CHIKV) (pJM6-3-CHIKV 181/25-mkate) (D), and Middle East respiratory syndrome coronavirus (MERS) (GenBank accession no. KJ614529.1 ) (E). Nucleotide coverage depth (NT coverage) is indicated on the y axis, and nucleotide (NT) position is indicated on the x axis. The genome length for each virus is indicated on the x axis, and the percentage of the genome covered greater than 3 nucleotides is indicated. For FMDV, WNV, HRV-16, and CHIKV, data represent material from a single barcode. For MERS, the data shown is a combination of four barcodes generated from the same sample.

    Techniques Used: Sequencing, Generated

    The SOP can detect WNV infection in vitro and in vivo . (A and B) Infected WNV cells were spiked into uninfected cells (A) or uninfected tissues (B), libraries were prepared on RNA according to the SOP, and the libraries were examined by Illumina MiSeq. (C and D) Footpad (C) and brain tissue (D) from WNV-infected mice were analyzed at 5, 10, and 29 days postinfection for WNV-specific sequence reads by Illumina HiSeq. (A) Data representing the ability of the SOP to identify WNV-specific reads from limiting dilutions of WNV-infected Vero cells spiked into uninfected 293T cells. Mapped and unmapped reads from each sample are displayed. (B) The SOP identifies WNV-specific reads from limiting dilutions of WNV-infected Vero cells spiked into uninfected mouse tissues (spleen and brain). Mapped and unmapped reads from each sample are shown. (C) WNV was detected in the footpad RNA of mice prepared according to the SOP at the indicated times postinfection. (D) WNV-specific reads can be detected from brain tissue RNA of mice at the indicated times postinfection. For panels C and D, three mice per group were analyzed, and WNV-mapped and unmapped reads are shown.
    Figure Legend Snippet: The SOP can detect WNV infection in vitro and in vivo . (A and B) Infected WNV cells were spiked into uninfected cells (A) or uninfected tissues (B), libraries were prepared on RNA according to the SOP, and the libraries were examined by Illumina MiSeq. (C and D) Footpad (C) and brain tissue (D) from WNV-infected mice were analyzed at 5, 10, and 29 days postinfection for WNV-specific sequence reads by Illumina HiSeq. (A) Data representing the ability of the SOP to identify WNV-specific reads from limiting dilutions of WNV-infected Vero cells spiked into uninfected 293T cells. Mapped and unmapped reads from each sample are displayed. (B) The SOP identifies WNV-specific reads from limiting dilutions of WNV-infected Vero cells spiked into uninfected mouse tissues (spleen and brain). Mapped and unmapped reads from each sample are shown. (C) WNV was detected in the footpad RNA of mice prepared according to the SOP at the indicated times postinfection. (D) WNV-specific reads can be detected from brain tissue RNA of mice at the indicated times postinfection. For panels C and D, three mice per group were analyzed, and WNV-mapped and unmapped reads are shown.

    Techniques Used: Infection, In Vitro, In Vivo, Mouse Assay, Sequencing

    Defining the sensitivity of the SOP on Illumina MiSeq and HiSeq platforms. RNA from serial 10-fold dilutions of an HRV-16 virion stock was treated according to the SOP. Samples were pooled and sequenced on an Illumina MiSeq or HiSeq platform. The left y axis denotes the number of reads mapped to the HRV-16 reference genome, the right y axis denotes the percentage of the reference genome covered, and the x axis denotes the input PFU for each reaction. The solid black line demonstrates that sequencing reads were detected between 1 and 10 PFU on the MiSeq platform. A similar sensitivity is obtained on the HiSeq platform, as denoted by the solid red line. The corresponding percentage of the HRV-16 genomic coverage from each platform is denoted by a dashed black line (MiSeq) and a dashed red line (HiSeq). The slight enhancement of genomic coverage on the MiSeq platform, despite the fewer number of sequence reads, results from the longer read length on the MiSeq platform (300 nucleotides [nt]) over the HiSeq platform (100 nt), as sequencing capacity is in excess at all dilutions.
    Figure Legend Snippet: Defining the sensitivity of the SOP on Illumina MiSeq and HiSeq platforms. RNA from serial 10-fold dilutions of an HRV-16 virion stock was treated according to the SOP. Samples were pooled and sequenced on an Illumina MiSeq or HiSeq platform. The left y axis denotes the number of reads mapped to the HRV-16 reference genome, the right y axis denotes the percentage of the reference genome covered, and the x axis denotes the input PFU for each reaction. The solid black line demonstrates that sequencing reads were detected between 1 and 10 PFU on the MiSeq platform. A similar sensitivity is obtained on the HiSeq platform, as denoted by the solid red line. The corresponding percentage of the HRV-16 genomic coverage from each platform is denoted by a dashed black line (MiSeq) and a dashed red line (HiSeq). The slight enhancement of genomic coverage on the MiSeq platform, despite the fewer number of sequence reads, results from the longer read length on the MiSeq platform (300 nucleotides [nt]) over the HiSeq platform (100 nt), as sequencing capacity is in excess at all dilutions.

    Techniques Used: Sequencing

    NGS on SOP-generated HRV-16-specific sequence from pure and mixed samples is slightly less sensitive than quantitative real-time RT-PCR (qrRT-PCR). Four independent tests were conducted to determine the sensitivity of the SOP. Test 1 detects HRV-16 sequence from dilutions of purified virus. Test 2 detects HRV-16 sequence from dilutions of genomic RNA. Test 3 detects HRV-16 sequence from dilutions of virus spiked into H1 HeLa cells. Test 4 detects HRV-16 sequence from genomic RNA dilutions spiked into total HeLa cell RNA. A ribosomal removal step was performed for tests 3 and 4 prior to the initiation of the SOP. For each sample, a fraction of the RNA used to initiate the SOP was subjected to qrRT-PCR analysis. (A to D) HRV-16-specific reads obtained by MiSeq (black solid lines) are plotted on the left y axis and the cycle threshold (Ct) values are plotted on the right y axis (red lines). Sequencing reads not mapping to the HRV-16 reference are also indicated (black dashed lines). Corresponding HRV-16 input PFU values are plotted on the x axis. (A) The limit of detection (LOD) for test 1 in this experiment is between 10 1 and 10 2 input PFU. The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU). (B) The LOD for test 2 in this experiment is between 10 1 and 10 2 input PFU. The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU). (C) The LOD for test 3 in this experiment is between 10 2 and 10 3 input PFU. The corresponding LOD by qrRT-PCR is approximately 100-fold greater (10 0 to 10 1 input PFU). (D) The LOD for test 4 in this experiment is between 10 1 and 10 2 input PFU; however, single reads are detected down to an input of 10 −1 . The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU) when individual HRV-16 reads are not considered and approximately 10-fold less sensitive when individual HRV-16 reads are considered.
    Figure Legend Snippet: NGS on SOP-generated HRV-16-specific sequence from pure and mixed samples is slightly less sensitive than quantitative real-time RT-PCR (qrRT-PCR). Four independent tests were conducted to determine the sensitivity of the SOP. Test 1 detects HRV-16 sequence from dilutions of purified virus. Test 2 detects HRV-16 sequence from dilutions of genomic RNA. Test 3 detects HRV-16 sequence from dilutions of virus spiked into H1 HeLa cells. Test 4 detects HRV-16 sequence from genomic RNA dilutions spiked into total HeLa cell RNA. A ribosomal removal step was performed for tests 3 and 4 prior to the initiation of the SOP. For each sample, a fraction of the RNA used to initiate the SOP was subjected to qrRT-PCR analysis. (A to D) HRV-16-specific reads obtained by MiSeq (black solid lines) are plotted on the left y axis and the cycle threshold (Ct) values are plotted on the right y axis (red lines). Sequencing reads not mapping to the HRV-16 reference are also indicated (black dashed lines). Corresponding HRV-16 input PFU values are plotted on the x axis. (A) The limit of detection (LOD) for test 1 in this experiment is between 10 1 and 10 2 input PFU. The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU). (B) The LOD for test 2 in this experiment is between 10 1 and 10 2 input PFU. The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU). (C) The LOD for test 3 in this experiment is between 10 2 and 10 3 input PFU. The corresponding LOD by qrRT-PCR is approximately 100-fold greater (10 0 to 10 1 input PFU). (D) The LOD for test 4 in this experiment is between 10 1 and 10 2 input PFU; however, single reads are detected down to an input of 10 −1 . The corresponding LOD by qrRT-PCR is approximately 10-fold greater (10 0 to 10 1 input PFU) when individual HRV-16 reads are not considered and approximately 10-fold less sensitive when individual HRV-16 reads are considered.

    Techniques Used: Next-Generation Sequencing, Generated, Sequencing, Quantitative RT-PCR, Polymerase Chain Reaction, Purification

    19) Product Images from "Evaluation of metagenetic community analysis of planktonic copepods using Illumina MiSeq: Comparisons with morphological classification and metagenetic analysis using Roche 454"

    Article Title: Evaluation of metagenetic community analysis of planktonic copepods using Illumina MiSeq: Comparisons with morphological classification and metagenetic analysis using Roche 454

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181452

    Total species and MOTU numbers of copepods in field-collected samples. MOTU numbers were calculated at 97 and 99% similarity thresholds in metagenetic analyses using both Illumina MiSeq and Roche 454 data.
    Figure Legend Snippet: Total species and MOTU numbers of copepods in field-collected samples. MOTU numbers were calculated at 97 and 99% similarity thresholds in metagenetic analyses using both Illumina MiSeq and Roche 454 data.

    Techniques Used:

    Order-level taxonomic compositions of copepods in field-collected samples between (A) species and MOTUs, and (B) biomass and sequence reads. Metagenetic analyses using both Illumina MiSeq and Roche 454 data were performed at a 97% similarity threshold.
    Figure Legend Snippet: Order-level taxonomic compositions of copepods in field-collected samples between (A) species and MOTUs, and (B) biomass and sequence reads. Metagenetic analyses using both Illumina MiSeq and Roche 454 data were performed at a 97% similarity threshold.

    Techniques Used: Sequencing

    Family-level taxonomic compositions of calanoid copepods in field-collected samples between (A) species and MOTUs, and (B) biomass and sequence reads. Metagenetic analyses using both Illumina MiSeq and Roche 454 data were performed at a 97% similarity threshold.
    Figure Legend Snippet: Family-level taxonomic compositions of calanoid copepods in field-collected samples between (A) species and MOTUs, and (B) biomass and sequence reads. Metagenetic analyses using both Illumina MiSeq and Roche 454 data were performed at a 97% similarity threshold.

    Techniques Used: Sequencing

    MOTU numbers at different minimum abundance thresholds of sequence reads: Comparisons of mock community analysis using (A) Illumina MiSeq and (B) Roche 454, and analyses of field-collected samples using (C) Illumina MiSeq and (D) Roche 454. A 0.05% of total reads for a single sample is indicated by dashed lines. Effects of the abundance threshold on MOTU numbers were evaluated both at a 97 and 99% similarity threshold for MOTU clustering.
    Figure Legend Snippet: MOTU numbers at different minimum abundance thresholds of sequence reads: Comparisons of mock community analysis using (A) Illumina MiSeq and (B) Roche 454, and analyses of field-collected samples using (C) Illumina MiSeq and (D) Roche 454. A 0.05% of total reads for a single sample is indicated by dashed lines. Effects of the abundance threshold on MOTU numbers were evaluated both at a 97 and 99% similarity threshold for MOTU clustering.

    Techniques Used: Sequencing

    20) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation, Gas Chromatography

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).
    Figure Legend Snippet: Coverage of PR8 virus genome with the optimized RT-PCR protocol. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp) using two different fragmentation methods: Nextera XT transposase-based fragmentation (black lines) and mechanical Covaris shearing followed by adaptor ligation (orange lines). The obtained sequences were mapped to the reference genome (based on the plasmids used to generate the virus).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Ligation

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Related Articles

    Hybridization:

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water
    Article Snippet: The pooled library was diluted to a final concentration of 5 pM using pre-chilled hybridization buffer (HT1) (Illumina, Irvine, CA, USA) measured by Qubit 2.0 Fluorometer (Life technologies, Ottawa, ON, Canada). .. The 150 paired-end sequencing reaction was performed on a MiSeq Illumina platform at the Microbiome Laboratory (Department of Animal Science, University of Manitoba, Canada).

    Amplification:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Nevertheless, the average quality (Phred score) of the detected variants was higher on the Illumina MiSeq than on the Ion Torrent PGM, making the variants detected on the Illumina MiSeq more reliable. .. The number of false positive variants can be further reduced by cross-platform replication, but the different biases of the sequencing platforms may cause many true variants to be overlooked when cross-platform replicates are compared [ , ].

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water
    Article Snippet: In the final step, 15% of PhiX control library was spiked into the amplicon library to improve the unbalanced and biased base composition. .. The 150 paired-end sequencing reaction was performed on a MiSeq Illumina platform at the Microbiome Laboratory (Department of Animal Science, University of Manitoba, Canada).

    Article Title: The effects of sequencing platforms on phylogenetic resolution in 16 S rRNA gene profiling of human feces
    Article Snippet: Comparative analysis of GS FLX+-, MiSeq-, and PacBio-generated datasets demonstrated that the Illumina MiSeq exhibited high performance in terms of reflecting the shotgun data, and sequencing coverage. .. Comparative analysis of GS FLX+-, MiSeq-, and PacBio-generated datasets demonstrated that the Illumina MiSeq exhibited high performance in terms of reflecting the shotgun data, and sequencing coverage.

    Sample Prep:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: For clinical samples, the shorter turnaround time of the Ion Torrent PGM (sample preparation, sequencing and analysis in about 2 days) is clearly advantageous to the Illumina MiSeq (about 3 days). .. In contrast, when analyzing many viral samples at high coverage, the greater output of the Illumina MiSeq is an important advantage.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: The proposed RT-PCR protocol and subsequent analysis pipeline for influenza viruses is widely applicable, e.g. to study vaccine composition, analyze virus evolution under selection pressure, monitor mutations associated with antiviral resistance, and assemble the reference genome of new viral isolates. .. For clinical samples, the shorter turnaround time of the Ion Torrent PGM (sample preparation, sequencing and analysis in about 2 days) is clearly advantageous to the Illumina MiSeq (about 3 days). .. In contrast, when analyzing many viral samples at high coverage, the greater output of the Illumina MiSeq is an important advantage.

    In Vitro:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Next, we determined the number of variants at each nucleotide position in the virus-derived sequences, which would reflect the quasispecies diversity of in vitro grown PR8 and PR8mut virus. .. Most variants were present in both sequencing duplicates, with the highest proportion of shared variants on the Illumina MiSeq (Table ).

    Plasmid Preparation:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: However, for plasmid DNA analysis the substitution error rate on the Ion Torrent PGM appeared to be lower than that of Illumina MiSeq (Figure ). .. Nevertheless, the average quality (Phred score) of the detected variants was higher on the Illumina MiSeq than on the Ion Torrent PGM, making the variants detected on the Illumina MiSeq more reliable.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Nevertheless, the average quality (average Phred score) of the tracer mutations was higher on the Illumina MiSeq (37.97 ± 0.09) than on the Ion Torrent PGM (30.72 ± 1.07), making the detected variants on the Illumina MiSeq more reliable. .. Nevertheless, the average quality (average Phred score) of the tracer mutations was higher on the Illumina MiSeq (37.97 ± 0.09) than on the Ion Torrent PGM (30.72 ± 1.07), making the detected variants on the Illumina MiSeq more reliable.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Plasmid samples were fragmented with Nextera XT transposase for Illumina MiSeq and mechanically sheared by Covaris, followed by adaptor ligation before Ion Torrent PGM sequencing. .. Nearly 70% of the unprocessed reads obtained on the Illumina MiSeq (2x250 bp sequencing) have a length of 250 bp, and the mean read length is 233.70 bp ± 1.65 bp (Figure A).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: The largest fluctuation was seen for the Illumina MiSeq (Figure B). .. The largest fluctuation was seen for the Illumina MiSeq (Figure B).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Most variants were present in both sequencing duplicates, with the highest proportion of shared variants on the Illumina MiSeq (Table ). .. Most variants were present in both sequencing duplicates, with the highest proportion of shared variants on the Illumina MiSeq (Table ).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: For the Illumina MiSeq, substitutions are the dominant error type with A-to-C and T-to-G being the most prevalent (Figure A), which is consistent with an earlier report [ ]. .. For the Illumina MiSeq, substitutions are the dominant error type with A-to-C and T-to-G being the most prevalent (Figure A), which is consistent with an earlier report [ ].

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: The per-base quality distribution on both sequencers, using the plasmid samples as template, is shown in Figure . .. For the processed reads obtained on the Illumina MiSeq, the 25th percentile of the Phred scores is ≥ 33 until position 251, and thus most of the sequencing reads are without sequencing error (Figure C).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: After this quality control, the sequencing reads were mapped to the reference sequence, resulting in a higher percentage of mapped reads for the Illumina MiSeq. .. After this quality control, the sequencing reads were mapped to the reference sequence, resulting in a higher percentage of mapped reads for the Illumina MiSeq.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: For the Illumina MiSeq, the broken pairs resulting from trimming and filtering were also removed. .. For the Illumina MiSeq, the broken pairs resulting from trimming and filtering were also removed.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Paragraph title: High-throughput sequencing of plasmid samples ... We selected the Illumina MiSeq and the Ion Torrent PGM sequencing platforms because the accuracy of single nucleotide polymorphism (SNP) identification of these two popular NGS platforms is unclear.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: As for the plasmid samples, all of the indels in the samples sequenced on the Illumina MiSeq and most of the indels in the samples sequenced on the Ion Torrent PGM were present in homopolymer regions. .. Nearly all SNPs were detected with a higher average Phred score on the Illumina MiSeq (37.39 ± 0.43 for PR8) and were thus more reliable than on the Ion Torrent PGM (28.58 ± 2.44 for PR8).

    Ligation:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Plasmid samples were fragmented with Nextera XT transposase for Illumina MiSeq and mechanically sheared by Covaris, followed by adaptor ligation before Ion Torrent PGM sequencing. .. Nearly 70% of the unprocessed reads obtained on the Illumina MiSeq (2x250 bp sequencing) have a length of 250 bp, and the mean read length is 233.70 bp ± 1.65 bp (Figure A).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: In contrast, when analyzing many viral samples at high coverage, the greater output of the Illumina MiSeq is an important advantage. .. In contrast, when analyzing many viral samples at high coverage, the greater output of the Illumina MiSeq is an important advantage.

    Mutagenesis:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: We considered the frequency of a given nucleotide significant (a real mutation) when it was higher than twice the sequencing error background, i.e. above 0.16% for the Illumina MiSeq and above 0.24% for the Ion Torrent PGM. .. In contrast, the variant calls on the Illumina MiSeq were mainly SNPs (Table ).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: We selected the Illumina MiSeq and the Ion Torrent PGM sequencing platforms because the accuracy of single nucleotide polymorphism (SNP) identification of these two popular NGS platforms is unclear. .. We used plasmid DNA to compare the accuracy of the sequencing output because it is genetically very stable.

    Next-Generation Sequencing:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Our aim was twofold: (1) to compare the performance of two high-throughput sequencing instruments; (2) to determine the complexity of an influenza A virus quasispecies (to count the number of nucleotide variants present in a swarm of genomes of that virus). .. We selected the Illumina MiSeq and the Ion Torrent PGM sequencing platforms because the accuracy of single nucleotide polymorphism (SNP) identification of these two popular NGS platforms is unclear. .. A study by Quail and colleagues concluded that the overall SNP calling rate is slightly higher for the data generated by Ion Torrent PGM than for Illumina MiSeq data [ ], whereas Loman and colleagues reported a lower substitution error rate for the Illumina MiSeq [ ].

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: We selected the Illumina MiSeq and the Ion Torrent PGM sequencing platforms because the accuracy of single nucleotide polymorphism (SNP) identification of these two popular NGS platforms is unclear. .. A study by Quail and colleagues concluded that the overall SNP calling rate is slightly higher for the data generated by Ion Torrent PGM than for Illumina MiSeq data [ ], whereas Loman and colleagues reported a lower substitution error rate for the Illumina MiSeq [ ].

    Polymerase Chain Reaction:

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water
    Article Snippet: Next, PCR products were purified using ZR-96 DNA Clean-up Kit (ZYMO Research, Irvine, CA, USA) to remove primers, dNTPs and reaction components. .. The 150 paired-end sequencing reaction was performed on a MiSeq Illumina platform at the Microbiome Laboratory (Department of Animal Science, University of Manitoba, Canada).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Nearly 70% of the unprocessed reads obtained on the Illumina MiSeq (2x250 bp sequencing) have a length of 250 bp, and the mean read length is 233.70 bp ± 1.65 bp (Figure A). .. These values are lower than expected since the Ion PGM Template OT2 400 Kit, Ion PGM Sequencing 400 Kit and Ion 318 chip v2 (revision 2.0) that we used should offer sequence reads of 400 bp according to their manuals.

    Hemagglutination Assay:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Nearly all SNPs were detected with a higher average Phred score on the Illumina MiSeq (37.39 ± 0.43 for PR8) and were thus more reliable than on the Ion Torrent PGM (28.58 ± 2.44 for PR8). .. The average difference between the frequencies of a variant in PR8 sequencing duplicates was only 0.17% ± 0.12% for the Illumina MiSeq and 0.16% ± 0.18% for the Ion Torrent PGM, again indicating that both sequencing platforms provide reproducible output (Table ).

    Sequencing:

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water
    Article Snippet: For Illumina sequencing, customized sequencing primers for read1 (5′-TATGGTAATTGTGTGCCAGCMGCCGCGGTAA-3′), read2 (5′-AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT-3′) and index read (5′-ATTAGAWACCCBDGTAGTCCGGCTGACTGACT-3′), and mixture of sample and PhiX library were added to the MiSeq Reagent Kit V2 (300-cycle; Illumina, San Diego, CA, USA). .. The 150 paired-end sequencing reaction was performed on a MiSeq Illumina platform at the Microbiome Laboratory (Department of Animal Science, University of Manitoba, Canada). .. The sequence data are uploaded into the Sequence Read Archive (SRA) or NCBI ( http://www.ncbi.nlm.nih.gov/sra ) and accessible through accession number SRR2983316.

    Article Title: The effects of sequencing platforms on phylogenetic resolution in 16 S rRNA gene profiling of human feces
    Article Snippet: A summary of the error types (e.g., mismatch, insertion, and deletion) observed in each of the datasets is presented in terms of relative abundance in . .. Comparative analysis of GS FLX+-, MiSeq-, and PacBio-generated datasets demonstrated that the Illumina MiSeq exhibited high performance in terms of reflecting the shotgun data, and sequencing coverage. .. Accordingly, we focused our analysis on comparing three different amplification primer sets (V1–3, V3–4, and V4; n =165, for each dataset) generated by the Illumina MiSeq platform ( ).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Plasmid samples were fragmented with Nextera XT transposase for Illumina MiSeq and mechanically sheared by Covaris, followed by adaptor ligation before Ion Torrent PGM sequencing. .. Nearly 70% of the unprocessed reads obtained on the Illumina MiSeq (2x250 bp sequencing) have a length of 250 bp, and the mean read length is 233.70 bp ± 1.65 bp (Figure A). .. The length of the unprocessed reads generated by the Ion Torrent PGM (400-bp sequencing on Ion 318 chip v2) follows a Gaussian distribution with a peak around 280 bp and a mean read length of 261.06 bp ± 2.51 bp (Figure B).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Paragraph title: Mapping of the sequencing reads ... The largest fluctuation was seen for the Illumina MiSeq (Figure B).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: The number and types of nucleotide variants that were retained after applying the variant filter are presented in Table . .. Most variants were present in both sequencing duplicates, with the highest proportion of shared variants on the Illumina MiSeq (Table ). .. However, the variants that were identified in only one of the duplicates were actually also detectable in the duplicate sample, but just below one of the four variant filtering parameters.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: For clinical samples, the shorter turnaround time of the Ion Torrent PGM (sample preparation, sequencing and analysis in about 2 days) is clearly advantageous to the Illumina MiSeq (about 3 days). .. In contrast, when analyzing many viral samples at high coverage, the greater output of the Illumina MiSeq is an important advantage.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Bases with a Phred score of 30 (chance of a wrong base call of 1 in 1000) are a measure of high quality data. .. For the processed reads obtained on the Illumina MiSeq, the 25th percentile of the Phred scores is ≥ 33 until position 251, and thus most of the sequencing reads are without sequencing error (Figure C). .. For the reads obtained on the Ion Torrent PGM, the median of the Phred scores is ≥ 30 until position 266 (Figure D).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: It is known that Illumina MiSeq and Ion Torrent PGM sequencers perform rather poorly when sequencing DNA with very low or very high GC content, which leads to low sequencing coverage of AT and GC rich regions [ , ]. .. In addition, the Nextera transposon-based fragmentation that we used for the samples sequenced on the Illumina MiSeq has some sequence preference, which can lead to a fragmentation bias, particularly in small genomes [ ]. .. Since the plasmid reference sequence is known, we were confident that any mismatching nucleotide variant could be reported as a sequencing error.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Furthermore, the Phred score distribution across the reads, a measure of the intrinsic sequencing quality, was higher for the Illumina MiSeq data than for the Ion Torrent PGM data, resulting in a lower error rate. .. After this quality control, the sequencing reads were mapped to the reference sequence, resulting in a higher percentage of mapped reads for the Illumina MiSeq. .. The total mapping error rate of the Illumina MiSeq (mainly nucleotide substitutions) was lower than that of the Ion Torrent PGM (mainly indels).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Despite this stringent cut-off, false positive errors were still detected, mostly as a consequence of the sequence specific error profiles of both sequencers (Table , [ , , ]). .. In contrast, the variant calls on the Illumina MiSeq were mainly SNPs (Table ).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Our aim was twofold: (1) to compare the performance of two high-throughput sequencing instruments; (2) to determine the complexity of an influenza A virus quasispecies (to count the number of nucleotide variants present in a swarm of genomes of that virus). .. We selected the Illumina MiSeq and the Ion Torrent PGM sequencing platforms because the accuracy of single nucleotide polymorphism (SNP) identification of these two popular NGS platforms is unclear. .. A study by Quail and colleagues concluded that the overall SNP calling rate is slightly higher for the data generated by Ion Torrent PGM than for Illumina MiSeq data [ ], whereas Loman and colleagues reported a lower substitution error rate for the Illumina MiSeq [ ].

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: The frequencies of the sequencing variants detected by both sequencers in duplicate are presented in Tables (PR8) and 9 (PR8mut). .. Nearly all SNPs were detected with a higher average Phred score on the Illumina MiSeq (37.39 ± 0.43 for PR8) and were thus more reliable than on the Ion Torrent PGM (28.58 ± 2.44 for PR8).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: The proposed RT-PCR protocol and subsequent analysis pipeline for influenza viruses is widely applicable, e.g. to study vaccine composition, analyze virus evolution under selection pressure, monitor mutations associated with antiviral resistance, and assemble the reference genome of new viral isolates. .. For clinical samples, the shorter turnaround time of the Ion Torrent PGM (sample preparation, sequencing and analysis in about 2 days) is clearly advantageous to the Illumina MiSeq (about 3 days). .. In contrast, when analyzing many viral samples at high coverage, the greater output of the Illumina MiSeq is an important advantage.

    Concentration Assay:

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water
    Article Snippet: The pooled library was diluted to a final concentration of 5 pM using pre-chilled hybridization buffer (HT1) (Illumina, Irvine, CA, USA) measured by Qubit 2.0 Fluorometer (Life technologies, Ottawa, ON, Canada). .. The 150 paired-end sequencing reaction was performed on a MiSeq Illumina platform at the Microbiome Laboratory (Department of Animal Science, University of Manitoba, Canada).

    Generated:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Nevertheless, the average quality (Phred score) of the detected variants was higher on the Illumina MiSeq than on the Ion Torrent PGM, making the variants detected on the Illumina MiSeq more reliable. .. The number of false positive variants can be further reduced by cross-platform replication, but the different biases of the sequencing platforms may cause many true variants to be overlooked when cross-platform replicates are compared [ , ].

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water
    Article Snippet: The V4 library was then generated by pooling 200 ng of each sample, quantified by Picogreen dsDNA (Invitrogen, Burlington, NY, USA). .. The 150 paired-end sequencing reaction was performed on a MiSeq Illumina platform at the Microbiome Laboratory (Department of Animal Science, University of Manitoba, Canada).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Nearly 70% of the unprocessed reads obtained on the Illumina MiSeq (2x250 bp sequencing) have a length of 250 bp, and the mean read length is 233.70 bp ± 1.65 bp (Figure A). .. Nearly 70% of the unprocessed reads obtained on the Illumina MiSeq (2x250 bp sequencing) have a length of 250 bp, and the mean read length is 233.70 bp ± 1.65 bp (Figure A).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: We selected the Illumina MiSeq and the Ion Torrent PGM sequencing platforms because the accuracy of single nucleotide polymorphism (SNP) identification of these two popular NGS platforms is unclear. .. We used plasmid DNA to compare the accuracy of the sequencing output because it is genetically very stable.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: We selected the Illumina MiSeq and the Ion Torrent PGM sequencing platforms because the accuracy of single nucleotide polymorphism (SNP) identification of these two popular NGS platforms is unclear. .. A study by Quail and colleagues concluded that the overall SNP calling rate is slightly higher for the data generated by Ion Torrent PGM than for Illumina MiSeq data [ ], whereas Loman and colleagues reported a lower substitution error rate for the Illumina MiSeq [ ]. .. We used plasmid DNA to compare the accuracy of the sequencing output because it is genetically very stable.

    Selection:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Nearly 70% of the unprocessed reads obtained on the Illumina MiSeq (2x250 bp sequencing) have a length of 250 bp, and the mean read length is 233.70 bp ± 1.65 bp (Figure A). .. These values are lower than expected since the Ion PGM Template OT2 400 Kit, Ion PGM Sequencing 400 Kit and Ion 318 chip v2 (revision 2.0) that we used should offer sequence reads of 400 bp according to their manuals.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: The proposed RT-PCR protocol and subsequent analysis pipeline for influenza viruses is widely applicable, e.g. to study vaccine composition, analyze virus evolution under selection pressure, monitor mutations associated with antiviral resistance, and assemble the reference genome of new viral isolates. .. In contrast, when analyzing many viral samples at high coverage, the greater output of the Illumina MiSeq is an important advantage.

    High Throughput Screening Assay:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Paragraph title: High-throughput sequencing of plasmid samples ... We selected the Illumina MiSeq and the Ion Torrent PGM sequencing platforms because the accuracy of single nucleotide polymorphism (SNP) identification of these two popular NGS platforms is unclear.

    Modification:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Next, the sequencing reads were trimmed from both sides using the modified Mott trimming algorithm to reach a Q20 score, which means that the chance that a particular base in the sequence is called incorrectly by the sequencer is 1 in 100. .. For the Illumina MiSeq, the broken pairs resulting from trimming and filtering were also removed.

    Purification:

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water
    Article Snippet: Next, PCR products were purified using ZR-96 DNA Clean-up Kit (ZYMO Research, Irvine, CA, USA) to remove primers, dNTPs and reaction components. .. The 150 paired-end sequencing reaction was performed on a MiSeq Illumina platform at the Microbiome Laboratory (Department of Animal Science, University of Manitoba, Canada).

    Chromatin Immunoprecipitation:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Nearly 70% of the unprocessed reads obtained on the Illumina MiSeq (2x250 bp sequencing) have a length of 250 bp, and the mean read length is 233.70 bp ± 1.65 bp (Figure A). .. The length of the unprocessed reads generated by the Ion Torrent PGM (400-bp sequencing on Ion 318 chip v2) follows a Gaussian distribution with a peak around 280 bp and a mean read length of 261.06 bp ± 2.51 bp (Figure B).

    Gas Chromatography:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: It is known that Illumina MiSeq and Ion Torrent PGM sequencers perform rather poorly when sequencing DNA with very low or very high GC content, which leads to low sequencing coverage of AT and GC rich regions [ , ]. .. For the Illumina MiSeq, substitutions are the dominant error type with A-to-C and T-to-G being the most prevalent (Figure A), which is consistent with an earlier report [ ].

    Variant Assay:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: After variant calling, the resulting hits were filtered based on frequency, forward/reverse balance, average quality, and independent counts to remove false positive variants. .. Nevertheless, the average quality (Phred score) of the detected variants was higher on the Illumina MiSeq than on the Ion Torrent PGM, making the variants detected on the Illumina MiSeq more reliable.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Paragraph title: Variant detection ... Nevertheless, the average quality (average Phred score) of the tracer mutations was higher on the Illumina MiSeq (37.97 ± 0.09) than on the Ion Torrent PGM (30.72 ± 1.07), making the detected variants on the Illumina MiSeq more reliable.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: The largest fluctuation was seen for the Illumina MiSeq (Figure B). .. In addition, the Nextera transposon-based fragmentation that we used for the samples sequenced on the Illumina MiSeq has some sequence preference, which can lead to a fragmentation bias, particularly in small genomes [ ].

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Most variants were present in both sequencing duplicates, with the highest proportion of shared variants on the Illumina MiSeq (Table ). .. Most variants were present in both sequencing duplicates, with the highest proportion of shared variants on the Illumina MiSeq (Table ).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: For the Illumina MiSeq, substitutions are the dominant error type with A-to-C and T-to-G being the most prevalent (Figure A), which is consistent with an earlier report [ ]. .. For the Illumina MiSeq, substitutions are the dominant error type with A-to-C and T-to-G being the most prevalent (Figure A), which is consistent with an earlier report [ ].

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: After this quality control, the sequencing reads were mapped to the reference sequence, resulting in a higher percentage of mapped reads for the Illumina MiSeq. .. However, for plasmid DNA analysis the substitution error rate on the Ion Torrent PGM appeared to be lower than that of Illumina MiSeq (Figure ).

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: The largest number of variants was deduced from the Ion Torrent PGM data, and all of them were indels (Table ). .. In contrast, the variant calls on the Illumina MiSeq were mainly SNPs (Table ). .. To eliminate false positive variants, we applied extra in silico filtering parameters.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Nearly all SNPs were detected with a higher average Phred score on the Illumina MiSeq (37.39 ± 0.43 for PR8) and were thus more reliable than on the Ion Torrent PGM (28.58 ± 2.44 for PR8). .. Nearly all SNPs were detected with a higher average Phred score on the Illumina MiSeq (37.39 ± 0.43 for PR8) and were thus more reliable than on the Ion Torrent PGM (28.58 ± 2.44 for PR8).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: Nevertheless, the average quality (Phred score) of the detected variants was higher on the Illumina MiSeq than on the Ion Torrent PGM, making the variants detected on the Illumina MiSeq more reliable. .. We then applied the analysis pipeline outlined in Figure to PR8 and PR8mut virus, which were generated by a plasmid-based reverse genetics system and amplified in MDCK cells.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: The proposed RT-PCR protocol and subsequent analysis pipeline for influenza viruses is widely applicable, e.g. to study vaccine composition, analyze virus evolution under selection pressure, monitor mutations associated with antiviral resistance, and assemble the reference genome of new viral isolates. .. In contrast, when analyzing many viral samples at high coverage, the greater output of the Illumina MiSeq is an important advantage.

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: For the Illumina MiSeq, the broken pairs resulting from trimming and filtering were also removed. .. For the Illumina MiSeq, the broken pairs resulting from trimming and filtering were also removed.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Illumina Inc miseq data
    Sequencing coverage distribution of high and low coverage areas. Histograms of the coverage at each position following mapping of the <t>Illumina</t> <t>MiSeq</t> reads to the Quiver-trimmed HGAP2 assembly are plotted for those positions that Quiver identifies as well-supported and those that are poorly-supported. Many of the remaining poorly supported internal positions have low coverage.
    Miseq Data, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/miseq data/product/Illumina Inc
    Average 99 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    miseq data - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

    Image Search Results


    Sequencing coverage distribution of high and low coverage areas. Histograms of the coverage at each position following mapping of the Illumina MiSeq reads to the Quiver-trimmed HGAP2 assembly are plotted for those positions that Quiver identifies as well-supported and those that are poorly-supported. Many of the remaining poorly supported internal positions have low coverage.

    Journal: BMC Genomics

    Article Title: Single molecule sequencing and genome assembly of a clinical specimen of Loa loa, the causative agent of loiasis

    doi: 10.1186/1471-2164-15-788

    Figure Lengend Snippet: Sequencing coverage distribution of high and low coverage areas. Histograms of the coverage at each position following mapping of the Illumina MiSeq reads to the Quiver-trimmed HGAP2 assembly are plotted for those positions that Quiver identifies as well-supported and those that are poorly-supported. Many of the remaining poorly supported internal positions have low coverage.

    Article Snippet: The sequencing reads were assembled with six different strategies: (1) Celera assembler (CA) [ ] with only Illumina MiSeq data; (2) MaSuRCA assembler [ ] with only Illumina MiSeq data; (3) CLC assembler (CLC bio, Cambridge, MA, USA) with only Illumina MiSeq data; (4) CA with PacBio reads corrected with Illumina MiSeq data [ ]; (5) CA with PacBio reads corrected with Illumina MiSeq data generated here and the Roche 454 data previously published [ ], and (6) HGAP2 with only the PacBio reads [ ].

    Techniques: Sequencing

    Assembly differences. The differences between the HGAP2 assembly presented here and the prior 454-based assembly are examined using NUCMER and BLASTN. The larger number of sequences uniquely in the HGAP2 assembly and the larger number of low complexity sequences suggests that the PacBio sequence data resolves more of the genome by spanning low complexity repeats. Some regions were identified in the 454-based assembly that were in the Illumina MiSeq assembly, suggesting that the sequences are missing from the HGAP2 assembly.

    Journal: BMC Genomics

    Article Title: Single molecule sequencing and genome assembly of a clinical specimen of Loa loa, the causative agent of loiasis

    doi: 10.1186/1471-2164-15-788

    Figure Lengend Snippet: Assembly differences. The differences between the HGAP2 assembly presented here and the prior 454-based assembly are examined using NUCMER and BLASTN. The larger number of sequences uniquely in the HGAP2 assembly and the larger number of low complexity sequences suggests that the PacBio sequence data resolves more of the genome by spanning low complexity repeats. Some regions were identified in the 454-based assembly that were in the Illumina MiSeq assembly, suggesting that the sequences are missing from the HGAP2 assembly.

    Article Snippet: The sequencing reads were assembled with six different strategies: (1) Celera assembler (CA) [ ] with only Illumina MiSeq data; (2) MaSuRCA assembler [ ] with only Illumina MiSeq data; (3) CLC assembler (CLC bio, Cambridge, MA, USA) with only Illumina MiSeq data; (4) CA with PacBio reads corrected with Illumina MiSeq data [ ]; (5) CA with PacBio reads corrected with Illumina MiSeq data generated here and the Roche 454 data previously published [ ], and (6) HGAP2 with only the PacBio reads [ ].

    Techniques: Sequencing

    Sequencing coverage distribution. A histogram of the coverage at each position following mapping of the Illumina MiSeq reads to all assemblies are plotted with log-transformed axes. Regions with abnormally little coverage exist, as well as those with excess coverage, for all assemblies. The peak in coverage for the Illumina data was at 51× coverage. In addition to this major peak, two additional peaks were identified at 200× and 450× in the HGAP2 assembly, reflecting collapsed repeats in the assembly including the mitochondrial genome, a L. loa interspersed repeat, and the rRNA.

    Journal: BMC Genomics

    Article Title: Single molecule sequencing and genome assembly of a clinical specimen of Loa loa, the causative agent of loiasis

    doi: 10.1186/1471-2164-15-788

    Figure Lengend Snippet: Sequencing coverage distribution. A histogram of the coverage at each position following mapping of the Illumina MiSeq reads to all assemblies are plotted with log-transformed axes. Regions with abnormally little coverage exist, as well as those with excess coverage, for all assemblies. The peak in coverage for the Illumina data was at 51× coverage. In addition to this major peak, two additional peaks were identified at 200× and 450× in the HGAP2 assembly, reflecting collapsed repeats in the assembly including the mitochondrial genome, a L. loa interspersed repeat, and the rRNA.

    Article Snippet: The sequencing reads were assembled with six different strategies: (1) Celera assembler (CA) [ ] with only Illumina MiSeq data; (2) MaSuRCA assembler [ ] with only Illumina MiSeq data; (3) CLC assembler (CLC bio, Cambridge, MA, USA) with only Illumina MiSeq data; (4) CA with PacBio reads corrected with Illumina MiSeq data [ ]; (5) CA with PacBio reads corrected with Illumina MiSeq data generated here and the Roche 454 data previously published [ ], and (6) HGAP2 with only the PacBio reads [ ].

    Techniques: Sequencing, Transformation Assay