miseq platform  (Illumina Inc)

 
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    Miseq Reagent kit V2
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    MS102-2003
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    Structured Review

    Illumina Inc miseq platform
    Metagenomic identification of <t>CHIKV</t> and EBOV from clinical blood samples by nanopore sequencing. a Time line of sequencing runs on flow cell #1 with sample reloading, plotted as a function of elapsed time in hours since the start of flow cell sequencing. b Cumulative numbers of all sequenced reads ( black line ) and target viral reads ( red line ) from the Chik1 run ( left panel ) and Ebola1 run ( right panel ), plotted as a function of individual sequencing run time in minutes. c Taxonomic donut charts generated using the MetaPORE bioinformatics analysis pipeline from the Chik1 run ( left panel ) and Ebola1 run ( right panel ). The total number of reads analyzed is shown in the center of the donut. d Coverage plots generated in MetaPORE by mapping reads aligning to CHIKV ( left , Chik1 run) or EBOV ( right , Ebola1 run) to the closest matching reference genome ( (e) , asterisk ). A corresponding pairwise identity plot is also shown for CHIKV, for which there is sufficient coverage. e Whole-genome phylogeny of CHIKV. Representative CHIKV genome sequences from the Asian-Pacific clade, including the Puerto Rico PR-S6 strain recovered by nanopore and <t>MiSeq</t> sequencing, or all available 188 near-complete or complete CHIKV genomes ( inset ), are included. Branch lengths are drawn proportionally to the number of nucleotide substitutions per position, and support values are shown for each node. were was analyzed in MetaPORE on a 64-core Ubuntu Linux server using the June 2014 and January 2015 NT databases as the reference databases for the CHIKV and EBOV samples, respectively

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    Images

    1) Product Images from "Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis"

    Article Title: Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

    Journal: Genome Medicine

    doi: 10.1186/s13073-015-0220-9

    Metagenomic identification of CHIKV and EBOV from clinical blood samples by nanopore sequencing. a Time line of sequencing runs on flow cell #1 with sample reloading, plotted as a function of elapsed time in hours since the start of flow cell sequencing. b Cumulative numbers of all sequenced reads ( black line ) and target viral reads ( red line ) from the Chik1 run ( left panel ) and Ebola1 run ( right panel ), plotted as a function of individual sequencing run time in minutes. c Taxonomic donut charts generated using the MetaPORE bioinformatics analysis pipeline from the Chik1 run ( left panel ) and Ebola1 run ( right panel ). The total number of reads analyzed is shown in the center of the donut. d Coverage plots generated in MetaPORE by mapping reads aligning to CHIKV ( left , Chik1 run) or EBOV ( right , Ebola1 run) to the closest matching reference genome ( (e) , asterisk ). A corresponding pairwise identity plot is also shown for CHIKV, for which there is sufficient coverage. e Whole-genome phylogeny of CHIKV. Representative CHIKV genome sequences from the Asian-Pacific clade, including the Puerto Rico PR-S6 strain recovered by nanopore and MiSeq sequencing, or all available 188 near-complete or complete CHIKV genomes ( inset ), are included. Branch lengths are drawn proportionally to the number of nucleotide substitutions per position, and support values are shown for each node. were was analyzed in MetaPORE on a 64-core Ubuntu Linux server using the June 2014 and January 2015 NT databases as the reference databases for the CHIKV and EBOV samples, respectively
    Figure Legend Snippet: Metagenomic identification of CHIKV and EBOV from clinical blood samples by nanopore sequencing. a Time line of sequencing runs on flow cell #1 with sample reloading, plotted as a function of elapsed time in hours since the start of flow cell sequencing. b Cumulative numbers of all sequenced reads ( black line ) and target viral reads ( red line ) from the Chik1 run ( left panel ) and Ebola1 run ( right panel ), plotted as a function of individual sequencing run time in minutes. c Taxonomic donut charts generated using the MetaPORE bioinformatics analysis pipeline from the Chik1 run ( left panel ) and Ebola1 run ( right panel ). The total number of reads analyzed is shown in the center of the donut. d Coverage plots generated in MetaPORE by mapping reads aligning to CHIKV ( left , Chik1 run) or EBOV ( right , Ebola1 run) to the closest matching reference genome ( (e) , asterisk ). A corresponding pairwise identity plot is also shown for CHIKV, for which there is sufficient coverage. e Whole-genome phylogeny of CHIKV. Representative CHIKV genome sequences from the Asian-Pacific clade, including the Puerto Rico PR-S6 strain recovered by nanopore and MiSeq sequencing, or all available 188 near-complete or complete CHIKV genomes ( inset ), are included. Branch lengths are drawn proportionally to the number of nucleotide substitutions per position, and support values are shown for each node. were was analyzed in MetaPORE on a 64-core Ubuntu Linux server using the June 2014 and January 2015 NT databases as the reference databases for the CHIKV and EBOV samples, respectively

    Techniques Used: Nanopore Sequencing, Sequencing, Flow Cytometry, Generated

    MetaPORE analysis of Illumina MiSeq data from samples containing CHIKV and EBOV. Taxonomic donut charts were generated from Illumina MiSeq data corresponding to the Chik1 run ( a ) and Ebola1 run ( b ) using the MetaPORE bioinformatics analysis pipeline. The total number of MiSeq reads analyzed is shown in the center of the donut. Note that given computational time constraints, only a subset of reads (n = 100,000) was analyzed using MetaPORE. Coverage and pairwise identity plots were generated from MiSeq CHIKV reads from the Chik1 sample (248,677 of 3,235,099 reads, 7.7 %) ( c ), or EBOV reads from the Ebola1 sample (20,820 of 2,743,589 reads, 0.76 %) ( d ), identified using SURPI analysis and LASTZ mapping {Harris, 2007 #34} at an e-value of 10-5 to the closest matching reference genome. Data were analyzed in MetaPORE on a 64-core Ubuntu Linux server using the June 2014 and January 2015 NT databases as the reference databases for the CHIKV and EBOV samples, respectively.
    Figure Legend Snippet: MetaPORE analysis of Illumina MiSeq data from samples containing CHIKV and EBOV. Taxonomic donut charts were generated from Illumina MiSeq data corresponding to the Chik1 run ( a ) and Ebola1 run ( b ) using the MetaPORE bioinformatics analysis pipeline. The total number of MiSeq reads analyzed is shown in the center of the donut. Note that given computational time constraints, only a subset of reads (n = 100,000) was analyzed using MetaPORE. Coverage and pairwise identity plots were generated from MiSeq CHIKV reads from the Chik1 sample (248,677 of 3,235,099 reads, 7.7 %) ( c ), or EBOV reads from the Ebola1 sample (20,820 of 2,743,589 reads, 0.76 %) ( d ), identified using SURPI analysis and LASTZ mapping {Harris, 2007 #34} at an e-value of 10-5 to the closest matching reference genome. Data were analyzed in MetaPORE on a 64-core Ubuntu Linux server using the June 2014 and January 2015 NT databases as the reference databases for the CHIKV and EBOV samples, respectively.

    Techniques Used: Generated

    Metagenomic sequencing workflow for MinION nanopore sequencing compared to Illumina MiSeq sequencing. a Overall workflow. b Steps in the MetaPORE real-time analysis pipeline. The turnaround time for sample-to-detection nanopore sequencing, defined here as the cumulative time taken for nucleic acid extraction, reverse transcription, library preparation, sequencing, MetaPORE bioinformatics analysis, and pathogen detection, was under 6 hr, while Illumina sequencing took over 20 hr. The time differential is accounted for by increased times for library quantitation, sequencing, and bioinformatics analysis with the Illumina protocol. *Assumes a 12-hr 50-bp single-end MiSeq run of ~12–15 million reads, with 50 bp the minimum estimated read length needed for accurate pathogen identification. **Denotes estimated average SURPI bioinformatics analysis run length for MiSeq data [ 19 ]. The stopwatch is depicted as a 12-hr clock
    Figure Legend Snippet: Metagenomic sequencing workflow for MinION nanopore sequencing compared to Illumina MiSeq sequencing. a Overall workflow. b Steps in the MetaPORE real-time analysis pipeline. The turnaround time for sample-to-detection nanopore sequencing, defined here as the cumulative time taken for nucleic acid extraction, reverse transcription, library preparation, sequencing, MetaPORE bioinformatics analysis, and pathogen detection, was under 6 hr, while Illumina sequencing took over 20 hr. The time differential is accounted for by increased times for library quantitation, sequencing, and bioinformatics analysis with the Illumina protocol. *Assumes a 12-hr 50-bp single-end MiSeq run of ~12–15 million reads, with 50 bp the minimum estimated read length needed for accurate pathogen identification. **Denotes estimated average SURPI bioinformatics analysis run length for MiSeq data [ 19 ]. The stopwatch is depicted as a 12-hr clock

    Techniques Used: Sequencing, Nanopore Sequencing, Quantitation Assay

    Metagenomic identification of EBOV from a clinical blood sample by nanopore sequencing and MetaPORE real-time bioinformatics analysis. Nanopore data generated from the Ebola2 library and sequenced on flow cell #3 were analyzed in real time using the MetaPORE bioinformatics analysis pipeline, and compared to corresponding Illumina MiSeq data. a Time line of nanopore sequencing runs on flow cell #3 with sample reloading, plotted as a function of elapsed time in hours since the start of flow cell sequencing. b Cumulative numbers of all sequenced reads (black line) and target viral reads (red line) from the nanopore run (left panel) or MiSeq run (right panel), plotted as a function of individual sequencing run time in minutes. c Taxonomic donut charts generated by real-time MetaPORE analysis of the nanopore reads (left panel) and post-run analysis of the MiSeq reads (right panel). The total number of reads analyzed is shown in the center of the donut. Note that given computational time constraints, only a subset of MiSeq reads (n = 100,000) was analyzed using MetaPORE. d Coverage and pairwise identity plots generated from nanopore (left panel) or MiSeq data (right panel) by mapping reads aligning to EBOV to the closest matching reference genome ((e), asterisk). e Whole-genome phylogeny of EBOV. Representative EBOV genome sequences, including those from the 2014-2015 West Africa outbreak (tan) and 2014 DRC outbreak (pink), are included. Branch lengths are drawn proportionally to the number of nucleotide substitutions per position, and support values are shown for each node. Data were analyzed in MetaPORE on a 64-core Ubuntu Linux server using the January 2015 NT reference database.
    Figure Legend Snippet: Metagenomic identification of EBOV from a clinical blood sample by nanopore sequencing and MetaPORE real-time bioinformatics analysis. Nanopore data generated from the Ebola2 library and sequenced on flow cell #3 were analyzed in real time using the MetaPORE bioinformatics analysis pipeline, and compared to corresponding Illumina MiSeq data. a Time line of nanopore sequencing runs on flow cell #3 with sample reloading, plotted as a function of elapsed time in hours since the start of flow cell sequencing. b Cumulative numbers of all sequenced reads (black line) and target viral reads (red line) from the nanopore run (left panel) or MiSeq run (right panel), plotted as a function of individual sequencing run time in minutes. c Taxonomic donut charts generated by real-time MetaPORE analysis of the nanopore reads (left panel) and post-run analysis of the MiSeq reads (right panel). The total number of reads analyzed is shown in the center of the donut. Note that given computational time constraints, only a subset of MiSeq reads (n = 100,000) was analyzed using MetaPORE. d Coverage and pairwise identity plots generated from nanopore (left panel) or MiSeq data (right panel) by mapping reads aligning to EBOV to the closest matching reference genome ((e), asterisk). e Whole-genome phylogeny of EBOV. Representative EBOV genome sequences, including those from the 2014-2015 West Africa outbreak (tan) and 2014 DRC outbreak (pink), are included. Branch lengths are drawn proportionally to the number of nucleotide substitutions per position, and support values are shown for each node. Data were analyzed in MetaPORE on a 64-core Ubuntu Linux server using the January 2015 NT reference database.

    Techniques Used: Nanopore Sequencing, Generated, Flow Cytometry, Sequencing

    2) Product Images from "A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps"

    Article Title: A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064084

    Coverage depth from a sequencing run using an Illumina U-2 OS osteosarcoma cell line library prepared on the AMCC chip. The library was run on the MiSeq using the 2x150 bp paired-end sequencing protocol. (a) Distribution of sequencing reads across the reference human genome, which has been divided into 1 million bins to assess coverage uniformity. (b) Average coverage across different chromosomes. (c) Average sequencing depth across different chromosomes. The Y chromosome is absent from the U-2 OS osteosarcoma cell line.
    Figure Legend Snippet: Coverage depth from a sequencing run using an Illumina U-2 OS osteosarcoma cell line library prepared on the AMCC chip. The library was run on the MiSeq using the 2x150 bp paired-end sequencing protocol. (a) Distribution of sequencing reads across the reference human genome, which has been divided into 1 million bins to assess coverage uniformity. (b) Average coverage across different chromosomes. (c) Average sequencing depth across different chromosomes. The Y chromosome is absent from the U-2 OS osteosarcoma cell line.

    Techniques Used: Sequencing, Chromatin Immunoprecipitation

    Coverage depths from sequencing runs using E. coli strain DH10B libraries prepared on the AMCC chip. (a) Ion Torrent libraries were run on the Ion Torrent PGM using the 100 bp sequencing protocol. (b) Illumina libraries were run on the MiSeq using the 2x25 bp paired-end sequencing protocol. Libraries labeled
    Figure Legend Snippet: Coverage depths from sequencing runs using E. coli strain DH10B libraries prepared on the AMCC chip. (a) Ion Torrent libraries were run on the Ion Torrent PGM using the 100 bp sequencing protocol. (b) Illumina libraries were run on the MiSeq using the 2x25 bp paired-end sequencing protocol. Libraries labeled "Control" were prepared off-chip using the standard benchtop protocols recommended by each manufacturer. Sequencing runs with uniform coverage are expected to yield a Poisson distribution of coverage depths, indicated by the curves labeled "Theoretical limit".

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Labeling

    3) Product Images from "Pseudomonas-Specific NGS Assay Provides Insight Into Abundance and Dynamics of Pseudomonas Species Including P. aeruginosa in a Cooling Tower"

    Article Title: Pseudomonas-Specific NGS Assay Provides Insight Into Abundance and Dynamics of Pseudomonas Species Including P. aeruginosa in a Cooling Tower

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01958

    Pseudomonas community composition of cooling tower water samples assessed by genus-specific 16S rRNA gene amplicon sequencing with Illumina MiSeq. Data presented as bar chart showing relative abundance of phylotypes (%). Phylotypes can correspond to species or a cluster of species according to amplified fragment resolution. The species present in each cluster are described in Supplementary Figure 1 . Bar chart of data analyzed according to Pseudomonas phylogenetic species groups is shown in Supplementary Figure 6 . Samples collected monthly in 2013 and 2014 are separated by year with a dashed line. Bar designated as Overall presents mean relative abundance of Pseudomonas phylotypes for all samples studied. Jan, January; Feb, February; Mar, March; Apr, April; Jun, June; Jul, July; Oct, October; Nov, November; Dec, December.
    Figure Legend Snippet: Pseudomonas community composition of cooling tower water samples assessed by genus-specific 16S rRNA gene amplicon sequencing with Illumina MiSeq. Data presented as bar chart showing relative abundance of phylotypes (%). Phylotypes can correspond to species or a cluster of species according to amplified fragment resolution. The species present in each cluster are described in Supplementary Figure 1 . Bar chart of data analyzed according to Pseudomonas phylogenetic species groups is shown in Supplementary Figure 6 . Samples collected monthly in 2013 and 2014 are separated by year with a dashed line. Bar designated as Overall presents mean relative abundance of Pseudomonas phylotypes for all samples studied. Jan, January; Feb, February; Mar, March; Apr, April; Jun, June; Jul, July; Oct, October; Nov, November; Dec, December.

    Techniques Used: Amplification, Sequencing

    Error rate distribution. Analysis of the NGS library preparation, Illumina MiSeq amplification and sequencing using 15,000 reads of P. aeruginosa DSM 50071 T . Substitution (blue), unknown base (red), and deletion (green) rates are shown.
    Figure Legend Snippet: Error rate distribution. Analysis of the NGS library preparation, Illumina MiSeq amplification and sequencing using 15,000 reads of P. aeruginosa DSM 50071 T . Substitution (blue), unknown base (red), and deletion (green) rates are shown.

    Techniques Used: Next-Generation Sequencing, Amplification, Sequencing

    4) Product Images from "Dispersal of Bacillus subtilis and its effect on strawberry phyllosphere microbiota under open field and protection conditions"

    Article Title: Dispersal of Bacillus subtilis and its effect on strawberry phyllosphere microbiota under open field and protection conditions

    Journal: Scientific Reports

    doi: 10.1038/srep22611

    Principal component analysis of microbial communities on strawberry leaves. First two Principle Components derived from correlation in individual OTU abundance among individual samples for bacteria ( a ) and fungi ( b ) – the OTU abundance was calculated as log 2 (x i + 1)/∑(log 2 (x i + 1)) for a given sample where x is the original counts value for the i th OTU. The two clusters were derived from the weighted UniFrac distance using the Ward method. Strawberry plants of cv. Vibrant grown in the open or under protection were sprayed or not sprayed with Serenade (a commercial formulated product of Bacillus subtilis ); leaves were sampled 4 hours (4H) and 8 days (8D) after spraying. In addition, leaves emerged after spraying were also sampled on day 8. Microbial populations on the phyllosphere were profiled with the Illumina MiSeq.
    Figure Legend Snippet: Principal component analysis of microbial communities on strawberry leaves. First two Principle Components derived from correlation in individual OTU abundance among individual samples for bacteria ( a ) and fungi ( b ) – the OTU abundance was calculated as log 2 (x i + 1)/∑(log 2 (x i + 1)) for a given sample where x is the original counts value for the i th OTU. The two clusters were derived from the weighted UniFrac distance using the Ward method. Strawberry plants of cv. Vibrant grown in the open or under protection were sprayed or not sprayed with Serenade (a commercial formulated product of Bacillus subtilis ); leaves were sampled 4 hours (4H) and 8 days (8D) after spraying. In addition, leaves emerged after spraying were also sampled on day 8. Microbial populations on the phyllosphere were profiled with the Illumina MiSeq.

    Techniques Used: Derivative Assay

    Number of sequence reads of Bacillus subtilis (ln transformed) for all individual samples. Strawberry plants of cv. Vibrant grown in the open or under protection were sprayed with Serenade (a commercial formulated product of Bacillus subtilis ) or not (Control); leaves were sampled 4 hours (4H) and 8 days (8D) after spraying. In addition, leaves emerged after the spraying (New) were also sampled on day 8. The triangle and circle points represent two replicate experiments. Microbial populations on the phyllosphere were profiled with the Illumina MiSeq.
    Figure Legend Snippet: Number of sequence reads of Bacillus subtilis (ln transformed) for all individual samples. Strawberry plants of cv. Vibrant grown in the open or under protection were sprayed with Serenade (a commercial formulated product of Bacillus subtilis ) or not (Control); leaves were sampled 4 hours (4H) and 8 days (8D) after spraying. In addition, leaves emerged after the spraying (New) were also sampled on day 8. The triangle and circle points represent two replicate experiments. Microbial populations on the phyllosphere were profiled with the Illumina MiSeq.

    Techniques Used: Sequencing, Transformation Assay

    5) Product Images from "The effects of sequencing platforms on phylogenetic resolution in 16 S rRNA gene profiling of human feces"

    Article Title: The effects of sequencing platforms on phylogenetic resolution in 16 S rRNA gene profiling of human feces

    Journal: Scientific Data

    doi: 10.1038/sdata.2018.68

    Relationship between sequence read length and assignment to database. ( a and b ) Sequences ( n =10,000) covering the V1–4 (red), V1–3 (light blue), V3–4 (blue), V4 (dark blue), and V1–9 (green) regions of the 16 S rRNA gene were extracted in silico from the GG 16 S rRNA gene database. ( a ) Pairwise similarities between targeted regions are represented as similarity distributions (lower left), similarity histograms (diagonal), and correlation coefficient values (upper right). ( b ) Accuracy of phylogenetic reconstruction per targeted region. Precision (x-axis) and recall (y-axis) were calculated based on 100 randomly chosen bootstrap replications. ( c ) Distribution of sequence (30,000 sub-sampled) similarities, including and excluding indel errors, between data generated by GS FLX+, Illumina MiSeq, and PacBio, and data from publically available PacBio sequences.
    Figure Legend Snippet: Relationship between sequence read length and assignment to database. ( a and b ) Sequences ( n =10,000) covering the V1–4 (red), V1–3 (light blue), V3–4 (blue), V4 (dark blue), and V1–9 (green) regions of the 16 S rRNA gene were extracted in silico from the GG 16 S rRNA gene database. ( a ) Pairwise similarities between targeted regions are represented as similarity distributions (lower left), similarity histograms (diagonal), and correlation coefficient values (upper right). ( b ) Accuracy of phylogenetic reconstruction per targeted region. Precision (x-axis) and recall (y-axis) were calculated based on 100 randomly chosen bootstrap replications. ( c ) Distribution of sequence (30,000 sub-sampled) similarities, including and excluding indel errors, between data generated by GS FLX+, Illumina MiSeq, and PacBio, and data from publically available PacBio sequences.

    Techniques Used: Sequencing, In Silico, Generated

    Taxonomy profiles of the Illumina MiSeq datasets. The proportion of assigned sequences ( a ) and relative abundance of the assigned sequences ( b ) from the MiSeq V1–3 ( n =165, light blue), V3–4 ( n =165, blue), and V4 ( n =165, dark blue) datasets were determined by assigning sequences to the GG 16 S rRNA gene sequence database, and are represented at the family, genus, and species levels, respectively. Data were analyzed by ANOVA followed by Tukey’s post-hoc test (* P
    Figure Legend Snippet: Taxonomy profiles of the Illumina MiSeq datasets. The proportion of assigned sequences ( a ) and relative abundance of the assigned sequences ( b ) from the MiSeq V1–3 ( n =165, light blue), V3–4 ( n =165, blue), and V4 ( n =165, dark blue) datasets were determined by assigning sequences to the GG 16 S rRNA gene sequence database, and are represented at the family, genus, and species levels, respectively. Data were analyzed by ANOVA followed by Tukey’s post-hoc test (* P

    Techniques Used: Sequencing

    Profile of insertion and deletion errors within each platform-generated dataset. Plots of insertion and deletion abundance for ( a ) the GG 16 S rRNA gene database (self-comparison as a control) and the ( b ) GS FLX+, ( c ) Illumina MiSeq V1–3, ( d ) MiSeq V3–4, ( e ) MiSeq V4, and ( f ) PacBio datasets. The abundance of insertions (and deletions) was normalized against sequence length.
    Figure Legend Snippet: Profile of insertion and deletion errors within each platform-generated dataset. Plots of insertion and deletion abundance for ( a ) the GG 16 S rRNA gene database (self-comparison as a control) and the ( b ) GS FLX+, ( c ) Illumina MiSeq V1–3, ( d ) MiSeq V3–4, ( e ) MiSeq V4, and ( f ) PacBio datasets. The abundance of insertions (and deletions) was normalized against sequence length.

    Techniques Used: Generated, Sequencing

    Abundance patterns of bacterial taxa in Illumina MiSeq datasets. ( a ) The abundance patterns of bacterial taxa in the MiSeq V1–3 ( n =165, light blue), V3–4 ( n =165, blue), and V4 ( n =165, dark blue) datasets were analyzed according to linear discriminant analysis effect size (LEfSe). ( b ) The relative abundance of the families Ruminococcaceae and RF39 , and the genera Sphingomonas , Haemophilus , Methanobrevibacter , and Citrobacter in OTU tables for each dataset are represented as bar graphs. Data were analyzed by ANOVA, followed by Tukey’s post-hoc test (* P
    Figure Legend Snippet: Abundance patterns of bacterial taxa in Illumina MiSeq datasets. ( a ) The abundance patterns of bacterial taxa in the MiSeq V1–3 ( n =165, light blue), V3–4 ( n =165, blue), and V4 ( n =165, dark blue) datasets were analyzed according to linear discriminant analysis effect size (LEfSe). ( b ) The relative abundance of the families Ruminococcaceae and RF39 , and the genera Sphingomonas , Haemophilus , Methanobrevibacter , and Citrobacter in OTU tables for each dataset are represented as bar graphs. Data were analyzed by ANOVA, followed by Tukey’s post-hoc test (* P

    Techniques Used:

    Comparative analysis of human fecal bacterial NGS datasets. Fecal samples collected from 19 human subjects were sequenced using the indicated platforms: GS FLX+ (V1–4, red), Illumina MiSeq (V1–3, light blue; V3–4, blue; V4, dark blue), and PacBio CCS (V1–9, green). Whole-genome shotgun sequences generated by Illumina HiSeq (Shotgun 16 S, orange) were included as a reference for community structure without amplification bias. ( a ) The sequence data were clustered using a UPGMA dendrogram based on the Bray-Curtis dissimilarity matrix, and samples from the same individual are shown in the same color. The relative abundances of bacterial taxa are displayed as a heatmap over 27 families ( > 1% relative abundance). ( b ) The sequence data were clustered by principal component analysis.
    Figure Legend Snippet: Comparative analysis of human fecal bacterial NGS datasets. Fecal samples collected from 19 human subjects were sequenced using the indicated platforms: GS FLX+ (V1–4, red), Illumina MiSeq (V1–3, light blue; V3–4, blue; V4, dark blue), and PacBio CCS (V1–9, green). Whole-genome shotgun sequences generated by Illumina HiSeq (Shotgun 16 S, orange) were included as a reference for community structure without amplification bias. ( a ) The sequence data were clustered using a UPGMA dendrogram based on the Bray-Curtis dissimilarity matrix, and samples from the same individual are shown in the same color. The relative abundances of bacterial taxa are displayed as a heatmap over 27 families ( > 1% relative abundance). ( b ) The sequence data were clustered by principal component analysis.

    Techniques Used: Next-Generation Sequencing, Generated, Amplification, Sequencing

    6) Product Images from "Methodology challenges in studying human gut microbiota – effects of collection, storage, DNA extraction and next generation sequencing technologies"

    Article Title: Methodology challenges in studying human gut microbiota – effects of collection, storage, DNA extraction and next generation sequencing technologies

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23296-4

    Donor specificity is maintained independent of experimental pipeline. ( a ) PCA bi-plots of clr transformed data on Illumina MiSeq and Ion Torrent PGM sequencing platforms. ( b ) Relative abundance profiles of individual subjects on each platform for top 20 families. ( c ) Relative abundances per platform represented as mean ± SD, with R 2 values displayed from linear model fitted to log10 values.
    Figure Legend Snippet: Donor specificity is maintained independent of experimental pipeline. ( a ) PCA bi-plots of clr transformed data on Illumina MiSeq and Ion Torrent PGM sequencing platforms. ( b ) Relative abundance profiles of individual subjects on each platform for top 20 families. ( c ) Relative abundances per platform represented as mean ± SD, with R 2 values displayed from linear model fitted to log10 values.

    Techniques Used: Transformation Assay, Sequencing

    Annotated OTUs per platform at all taxonomic levels. Percentage (%) of annotated OTUs on each taxonomic level for both Illumina MiSeq (MiSeq) and Ion Torrent PGM (IT) sequencing platforms.
    Figure Legend Snippet: Annotated OTUs per platform at all taxonomic levels. Percentage (%) of annotated OTUs on each taxonomic level for both Illumina MiSeq (MiSeq) and Ion Torrent PGM (IT) sequencing platforms.

    Techniques Used: Sequencing

    7) Product Images from "Pseudomonas-Specific NGS Assay Provides Insight Into Abundance and Dynamics of Pseudomonas Species Including P. aeruginosa in a Cooling Tower"

    Article Title: Pseudomonas-Specific NGS Assay Provides Insight Into Abundance and Dynamics of Pseudomonas Species Including P. aeruginosa in a Cooling Tower

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01958

    Pseudomonas community composition of cooling tower water samples assessed by genus-specific 16S rRNA gene amplicon sequencing with Illumina MiSeq. Data presented as bar chart showing relative abundance of phylotypes (%). Phylotypes can correspond to species or a cluster of species according to amplified fragment resolution. The species present in each cluster are described in Supplementary Figure 1 . Bar chart of data analyzed according to Pseudomonas phylogenetic species groups is shown in Supplementary Figure 6 . Samples collected monthly in 2013 and 2014 are separated by year with a dashed line. Bar designated as Overall presents mean relative abundance of Pseudomonas phylotypes for all samples studied. Jan, January; Feb, February; Mar, March; Apr, April; Jun, June; Jul, July; Oct, October; Nov, November; Dec, December.
    Figure Legend Snippet: Pseudomonas community composition of cooling tower water samples assessed by genus-specific 16S rRNA gene amplicon sequencing with Illumina MiSeq. Data presented as bar chart showing relative abundance of phylotypes (%). Phylotypes can correspond to species or a cluster of species according to amplified fragment resolution. The species present in each cluster are described in Supplementary Figure 1 . Bar chart of data analyzed according to Pseudomonas phylogenetic species groups is shown in Supplementary Figure 6 . Samples collected monthly in 2013 and 2014 are separated by year with a dashed line. Bar designated as Overall presents mean relative abundance of Pseudomonas phylotypes for all samples studied. Jan, January; Feb, February; Mar, March; Apr, April; Jun, June; Jul, July; Oct, October; Nov, November; Dec, December.

    Techniques Used: Amplification, Sequencing

    Error rate distribution. Analysis of the NGS library preparation, Illumina MiSeq amplification and sequencing using 15,000 reads of P. aeruginosa DSM 50071 T . Substitution (blue), unknown base (red), and deletion (green) rates are shown.
    Figure Legend Snippet: Error rate distribution. Analysis of the NGS library preparation, Illumina MiSeq amplification and sequencing using 15,000 reads of P. aeruginosa DSM 50071 T . Substitution (blue), unknown base (red), and deletion (green) rates are shown.

    Techniques Used: Next-Generation Sequencing, Amplification, 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 "Identification of Associations between Bacterioplankton and Photosynthetic Picoeukaryotes in Coastal Waters"

    Article Title: Identification of Associations between Bacterioplankton and Photosynthetic Picoeukaryotes in Coastal Waters

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.00339

    Dendrograms based on Bray–Curtis similarity between Illumina MiSeq 16S rRNA gene libraries for (A) all OTUs, (B) non-Eukaryota OTUs .
    Figure Legend Snippet: Dendrograms based on Bray–Curtis similarity between Illumina MiSeq 16S rRNA gene libraries for (A) all OTUs, (B) non-Eukaryota OTUs .

    Techniques Used:

    Pie graphs showing the phylogenetic affiliations of non-Eukaryota 16S rRNA gene sequences in photosynthetic picoeukaryote sorts (P1, 1,000 cells), and bulk seawater (0.2–10 μm) from Illumina MiSeq libraries for each sampling date . The absolute numbers of sequences for the triplicate sort samples from each sampling date have been pooled. The colors for each phyla are indicated below the graphs.
    Figure Legend Snippet: Pie graphs showing the phylogenetic affiliations of non-Eukaryota 16S rRNA gene sequences in photosynthetic picoeukaryote sorts (P1, 1,000 cells), and bulk seawater (0.2–10 μm) from Illumina MiSeq libraries for each sampling date . The absolute numbers of sequences for the triplicate sort samples from each sampling date have been pooled. The colors for each phyla are indicated below the graphs.

    Techniques Used: Sampling

    Neighbor-joining phylogenetic tree of non-Eukaryota OTUs (97% similarity) in 16S rRNA gene Illumina MiSeq libraries with > 100 sequences present in photosynthetic picoeukaryote cell sorts (P1, 1,000 cells) . OTUs are indicated in bold and GenBank accession numbers for the closest relatives are written in square brackets. Bootstrap values > 50% (1,000 replicates) are indicated by circles with the size of the circles corresponding to the bootstrap value. A colored bar shows which phyla the branches of the tree are affiliated with. Heatmaps for photosynthetic picoeukaryote sort (P1, 1,000 cells) and seawater samples show the normalized number of sequences of each OTU in the different samples. The Synechococcus clade (18 OTUs, > 96% similarity) has been collapsed for clarity and is represented by denovo9700.
    Figure Legend Snippet: Neighbor-joining phylogenetic tree of non-Eukaryota OTUs (97% similarity) in 16S rRNA gene Illumina MiSeq libraries with > 100 sequences present in photosynthetic picoeukaryote cell sorts (P1, 1,000 cells) . OTUs are indicated in bold and GenBank accession numbers for the closest relatives are written in square brackets. Bootstrap values > 50% (1,000 replicates) are indicated by circles with the size of the circles corresponding to the bootstrap value. A colored bar shows which phyla the branches of the tree are affiliated with. Heatmaps for photosynthetic picoeukaryote sort (P1, 1,000 cells) and seawater samples show the normalized number of sequences of each OTU in the different samples. The Synechococcus clade (18 OTUs, > 96% similarity) has been collapsed for clarity and is represented by denovo9700.

    Techniques Used:

    10) Product Images from "Facile Discovery of a Diverse Panel of Anti-Ebola Virus Antibodies by Immune Repertoire Mining"

    Article Title: Facile Discovery of a Diverse Panel of Anti-Ebola Virus Antibodies by Immune Repertoire Mining

    Journal: Scientific Reports

    doi: 10.1038/srep13926

    Isolation of antibodies by mining the paired V H :V L repertoire of draining popliteal lymph node (PLN) antibody-secreting B cells. ( a ) Footpad immunization leads to a marked increase in cellularity within the ipsilateral popliteal lymph node relative to the contralateral lymph node (red and black arrows respectively). ( b ) PLN CD138 + cells isolated by magnetic sorting are deposited into 125 pL wells on PDMS slides that also contain poly(dT) beads. Cells are lysed in situ and mRNA is captured on the poly(dT) beads 33 . ( c ) The poly(dT) beads are emulsified and V H :V L amplicons are generated following reverse transcription and overlap extension PCR. ( d ) V H :V L amplicons are sequenced using Illumina 2 × 250 MiSeq and the highest frequency V H :V L pairs are identified via bioinformatics analysis. ( e ) Highest frequency V H (orange) and V L (green) genes are synthesized and cloned into IgH and IgL expression vectors containing human IgG1 (blue) and human kappa (yellow) constant regions, respectively. ( f ) Following co-transfection into Expi293 cells, recombinant IgG antibodies are expressed and purified.
    Figure Legend Snippet: Isolation of antibodies by mining the paired V H :V L repertoire of draining popliteal lymph node (PLN) antibody-secreting B cells. ( a ) Footpad immunization leads to a marked increase in cellularity within the ipsilateral popliteal lymph node relative to the contralateral lymph node (red and black arrows respectively). ( b ) PLN CD138 + cells isolated by magnetic sorting are deposited into 125 pL wells on PDMS slides that also contain poly(dT) beads. Cells are lysed in situ and mRNA is captured on the poly(dT) beads 33 . ( c ) The poly(dT) beads are emulsified and V H :V L amplicons are generated following reverse transcription and overlap extension PCR. ( d ) V H :V L amplicons are sequenced using Illumina 2 × 250 MiSeq and the highest frequency V H :V L pairs are identified via bioinformatics analysis. ( e ) Highest frequency V H (orange) and V L (green) genes are synthesized and cloned into IgH and IgL expression vectors containing human IgG1 (blue) and human kappa (yellow) constant regions, respectively. ( f ) Following co-transfection into Expi293 cells, recombinant IgG antibodies are expressed and purified.

    Techniques Used: Isolation, In Situ, Generated, Polymerase Chain Reaction, Synthesized, Clone Assay, Expressing, Cotransfection, Recombinant, Purification

    11) Product Images from "Linking T-cell receptor sequence to functional phenotype at the single-cell level"

    Article Title: Linking T-cell receptor sequence to functional phenotype at the single-cell level

    Journal:

    doi: 10.1038/nbt.2938

    Strategy for single-cell TCR sequencing and phenotyping, and determination of TCR-sequencing efficiency. ( a ) Strategy for simultaneous TCR-sequence determination and phenotyping of single, sorted T cells. Single T cells were sorted into 96-well plates. The initial RT-PCR reaction (reaction 1) uses 76 TCR primers and 34 phenotyping primers. An aliquot of the product of reaction 1 is used for two separate second nested PCR reactions (reaction 2), one for TCR sequencing and one for phenotyping. Using an aliquot of reaction 2 product as a template, a third PCR reaction is performed that incorporates individual barcodes into each well and enables subsequent sequencing using the Illumina MiSeq platform. For TCR sequencing, the third reaction can be split into separate TCRα and TCRβ reactions (for optimal efficiency), or the two TCR chains can be included in a single reaction. The products of reaction 3 are then combined and sequenced using the Illumina MiSeq platform. ( b,c ) Accuracy and efficiency of TCR sequencing using this method. ( b ) Strategy used to validate TCR sequencing. Into each 96-well test plate, individual human peripheral blood T cells were sorted into 80 wells (gray). Single Jurkat T cells were sorted into eight other wells (red), and the remaining eight wells (black) were left empty (blank). For sequencing of these test plates, reaction 3 was initially performed separately for TCRα and TCRβ (split). It was also repeated with TCRα and TCRβ amplified together in the same reaction (combined). ( c ) Efficiency of TCRα and TCRβ sequencing in split or combined formats. Plate 1 contained 80 single CD45RA+ CD4+ TCRαβ+ T cells, and plate 2 contained 80 single CD4+ or CD8+ TCRαβ+ T cells sorted from periphera blood of the same healthy human donor. Identical Jurkat sequences were obtained from all Jurkat wells. No sequences were obtained from any empty wells.
    Figure Legend Snippet: Strategy for single-cell TCR sequencing and phenotyping, and determination of TCR-sequencing efficiency. ( a ) Strategy for simultaneous TCR-sequence determination and phenotyping of single, sorted T cells. Single T cells were sorted into 96-well plates. The initial RT-PCR reaction (reaction 1) uses 76 TCR primers and 34 phenotyping primers. An aliquot of the product of reaction 1 is used for two separate second nested PCR reactions (reaction 2), one for TCR sequencing and one for phenotyping. Using an aliquot of reaction 2 product as a template, a third PCR reaction is performed that incorporates individual barcodes into each well and enables subsequent sequencing using the Illumina MiSeq platform. For TCR sequencing, the third reaction can be split into separate TCRα and TCRβ reactions (for optimal efficiency), or the two TCR chains can be included in a single reaction. The products of reaction 3 are then combined and sequenced using the Illumina MiSeq platform. ( b,c ) Accuracy and efficiency of TCR sequencing using this method. ( b ) Strategy used to validate TCR sequencing. Into each 96-well test plate, individual human peripheral blood T cells were sorted into 80 wells (gray). Single Jurkat T cells were sorted into eight other wells (red), and the remaining eight wells (black) were left empty (blank). For sequencing of these test plates, reaction 3 was initially performed separately for TCRα and TCRβ (split). It was also repeated with TCRα and TCRβ amplified together in the same reaction (combined). ( c ) Efficiency of TCRα and TCRβ sequencing in split or combined formats. Plate 1 contained 80 single CD45RA+ CD4+ TCRαβ+ T cells, and plate 2 contained 80 single CD4+ or CD8+ TCRαβ+ T cells sorted from periphera blood of the same healthy human donor. Identical Jurkat sequences were obtained from all Jurkat wells. No sequences were obtained from any empty wells.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Nested PCR, Polymerase Chain Reaction, Amplification

    12) Product Images from "Stationary and portable sequencing-based approaches for tracing wastewater contamination in urban stormwater systems"

    Article Title: Stationary and portable sequencing-based approaches for tracing wastewater contamination in urban stormwater systems

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29920-7

    Expenditures of time for assessing levels of human faecal contamination in water samples with different approaches. The first three bars demonstrate the amount of time needed with the experimental settings applied in this study, while the last two bars show the feasible time requirements of the Nanopore-based approach with settings optimised based on the results of this study. Computing time for sequence and data analysis (i.e., the steps after the actual sequencing) was calculated based on the computing power of 16 threads. The arrow under the MiSeq timeline indicates the starting point if only five samples had been processed. Time for the Colilert-18® test would not change regardless of sample size. The time for the barcoding procedure in the Nanopore library preparation was segmented from the library preparation hours and marked with “B”. The base-calling procedure for Nanopore can be conducted while the sequencing is ongoing. The optimised Nanopore timelines (the 4th and 5th bars) demonstrate the feasible time usage for assessing five water samples by using different barcoding approaches (ligation-based or transposase-based) with the sequencing depth of 1,000 reads per sample.
    Figure Legend Snippet: Expenditures of time for assessing levels of human faecal contamination in water samples with different approaches. The first three bars demonstrate the amount of time needed with the experimental settings applied in this study, while the last two bars show the feasible time requirements of the Nanopore-based approach with settings optimised based on the results of this study. Computing time for sequence and data analysis (i.e., the steps after the actual sequencing) was calculated based on the computing power of 16 threads. The arrow under the MiSeq timeline indicates the starting point if only five samples had been processed. Time for the Colilert-18® test would not change regardless of sample size. The time for the barcoding procedure in the Nanopore library preparation was segmented from the library preparation hours and marked with “B”. The base-calling procedure for Nanopore can be conducted while the sequencing is ongoing. The optimised Nanopore timelines (the 4th and 5th bars) demonstrate the feasible time usage for assessing five water samples by using different barcoding approaches (ligation-based or transposase-based) with the sequencing depth of 1,000 reads per sample.

    Techniques Used: Sequencing, Ligation

    13) Product Images from "Differential impact of transplantation on peripheral and tissue-associated viral reservoirs: Implications for HIV gene therapy"

    Article Title: Differential impact of transplantation on peripheral and tissue-associated viral reservoirs: Implications for HIV gene therapy

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006956

    ΔCCR5 cells persist in tissues of SHIV + animals. Tissues were collected longitudinally (figure panels [A-B] ) or at necropsy (panels [C-E] ) from transplanted animals, and the percentage of CCR5-edited alleles was quantified from total tissue homogenate by Illumina MiSeq. (A) Duodenum/Jejunum (Upper GI), colon (Lower GI) and peripheral lymph nodes from Group A animals transplanted prior to SHIV challenge. (B) Same as panel A, from Group B-C animals transplanted following SHIV infection and stable suppression by cART. (C) Necropsy tissues from Group A animals as in panel A. (D) Necropsy tissues from Group B animals in panel B that were necropsied following cART withdrawal. (E) Necropsy tissues from Group C animals in panel B that were necropsied while stably suppressed on cART.
    Figure Legend Snippet: ΔCCR5 cells persist in tissues of SHIV + animals. Tissues were collected longitudinally (figure panels [A-B] ) or at necropsy (panels [C-E] ) from transplanted animals, and the percentage of CCR5-edited alleles was quantified from total tissue homogenate by Illumina MiSeq. (A) Duodenum/Jejunum (Upper GI), colon (Lower GI) and peripheral lymph nodes from Group A animals transplanted prior to SHIV challenge. (B) Same as panel A, from Group B-C animals transplanted following SHIV infection and stable suppression by cART. (C) Necropsy tissues from Group A animals as in panel A. (D) Necropsy tissues from Group B animals in panel B that were necropsied following cART withdrawal. (E) Necropsy tissues from Group C animals in panel B that were necropsied while stably suppressed on cART.

    Techniques Used: Infection, Stable Transfection

    14) Product Images from "Ultrahigh-Throughput Multiplexing and Sequencing of > 500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform"

    Article Title: Ultrahigh-Throughput Multiplexing and Sequencing of > 500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform

    Journal: mSystems

    doi: 10.1128/mSystems.00029-19

    Heatmap of taxon relative abundances (rows) of samples (columns). Subject samples are separated by white lines, and samples are ordered by vaginal community state types and with the following color code: 1-step MiSeq, pink; 2-step HiSeq, blue; 2-step MiSeq, aqua. (B) tSNE representation of Jensen-Shannon distances between samples from the same subject. Samples primarily cluster by vaginal CST.
    Figure Legend Snippet: Heatmap of taxon relative abundances (rows) of samples (columns). Subject samples are separated by white lines, and samples are ordered by vaginal community state types and with the following color code: 1-step MiSeq, pink; 2-step HiSeq, blue; 2-step MiSeq, aqua. (B) tSNE representation of Jensen-Shannon distances between samples from the same subject. Samples primarily cluster by vaginal CST.

    Techniques Used:

    Forward and reverse read quality profiles for 300 cycles on the Illumina HiSeq (1,536 samples) and MiSeq (444 samples) platforms. Amplicon libraries were prepared using a 2-step PCR method. Shown for each cycle are the mean quality score (green line), the median quality score (solid purple line), and the quartiles of the quality score distribution (dotted purple lines).
    Figure Legend Snippet: Forward and reverse read quality profiles for 300 cycles on the Illumina HiSeq (1,536 samples) and MiSeq (444 samples) platforms. Amplicon libraries were prepared using a 2-step PCR method. Shown for each cycle are the mean quality score (green line), the median quality score (solid purple line), and the quartiles of the quality score distribution (dotted purple lines).

    Techniques Used: Amplification, Polymerase Chain Reaction

    15) Product Images from "Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity"

    Article Title: Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity

    Journal: Nature Communications

    doi: 10.1038/ncomms13642

    The IgDiscover iterative gene discovery process. ( a ) Following isolation of lymphocyte messenger RNA, an IgM library is constructed using either 5′RACE or multiplex PCR and sequenced using the Illumina MiSeq system. ( b ) Paired sequences are merged, and adapters are optionally removed. IgBLAST then assigns VH segments based on the starting database, and low-quality assignments are filtered (see Methods). ( c ) Windowed cluster analysis, (upper left panel), showing sequences assigned to a reference gene and binned in 2% windows to allow discrete consensus building. Linkage cluster analysis (upper right panel) of a subset of 300 sequences. Rows and columns of the matrix correspond to sequences. The colour at an intersection of a row and a column gives the number of differences between the corresponding sequences. The dendrograms (to the left and above, both identical) show the hierarchy found according to hierarchical clustering. Sequences are rearranged to conform to the clustering, putting similar sequences adjacent to each other. Clusters of similar sequences are visible as bright squares along the main diagonal. Colouring on the left indicates clusters detected by IgDiscover (one colour per cluster). Following consensus building, candidate germline sequences are processed using the pregermline filter, resulting in a new VH database. The assignment, clustering, consensus-building and pregermline filter steps are repeated for a set number of iterations using each new VH database for initial assignment. Examples of windowed cluster histograms (left two lower panels) and linkage cluster plots (right two lower panels) using databases containing newly identified candidate germline V genes. In the final iteration, the candidate V genes are processed using the germline filter to reveal the final database.
    Figure Legend Snippet: The IgDiscover iterative gene discovery process. ( a ) Following isolation of lymphocyte messenger RNA, an IgM library is constructed using either 5′RACE or multiplex PCR and sequenced using the Illumina MiSeq system. ( b ) Paired sequences are merged, and adapters are optionally removed. IgBLAST then assigns VH segments based on the starting database, and low-quality assignments are filtered (see Methods). ( c ) Windowed cluster analysis, (upper left panel), showing sequences assigned to a reference gene and binned in 2% windows to allow discrete consensus building. Linkage cluster analysis (upper right panel) of a subset of 300 sequences. Rows and columns of the matrix correspond to sequences. The colour at an intersection of a row and a column gives the number of differences between the corresponding sequences. The dendrograms (to the left and above, both identical) show the hierarchy found according to hierarchical clustering. Sequences are rearranged to conform to the clustering, putting similar sequences adjacent to each other. Clusters of similar sequences are visible as bright squares along the main diagonal. Colouring on the left indicates clusters detected by IgDiscover (one colour per cluster). Following consensus building, candidate germline sequences are processed using the pregermline filter, resulting in a new VH database. The assignment, clustering, consensus-building and pregermline filter steps are repeated for a set number of iterations using each new VH database for initial assignment. Examples of windowed cluster histograms (left two lower panels) and linkage cluster plots (right two lower panels) using databases containing newly identified candidate germline V genes. In the final iteration, the candidate V genes are processed using the germline filter to reveal the final database.

    Techniques Used: Isolation, Construct, Multiplex Assay, Polymerase Chain Reaction

    16) 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

    17) Product Images from "Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites"

    Article Title: Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites

    Journal: Human Gene Therapy Methods

    doi: 10.1089/hgtb.2015.060

    Illumina MiSeq MGS-PCR. Genomic DNA is sheared, resulting in smaller genomic fragments that may contain the vector provirus. Fragments are blunt ended and linker cassettes ligated to the ends of the fragment. Thirty rounds of exponential PCR are conducted
    Figure Legend Snippet: Illumina MiSeq MGS-PCR. Genomic DNA is sheared, resulting in smaller genomic fragments that may contain the vector provirus. Fragments are blunt ended and linker cassettes ligated to the ends of the fragment. Thirty rounds of exponential PCR are conducted

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation

    Illumina MiSeq universal primers. The Illumina universal primers (forward and reverse) consist of an adapter, indices, stem, and the user-defined specific primer sequences. In our adaptation for MGS-PCR, the forward primer is designed for the LTR, while
    Figure Legend Snippet: Illumina MiSeq universal primers. The Illumina universal primers (forward and reverse) consist of an adapter, indices, stem, and the user-defined specific primer sequences. In our adaptation for MGS-PCR, the forward primer is designed for the LTR, while

    Techniques Used: Polymerase Chain Reaction

    18) Product Images from "Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene"

    Article Title: Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene

    Journal: Scientific Reports

    doi: 10.1038/srep26314

    Sequence coverage of pHW-NS1(1-73)Dmd-GFP-NEP based on Illumina MiSeq sequencing. ( a ) Map of pHW-NS1(1-73)Dmd-GFP-NEP. ( b ) Sequence coverage (black line) and GC-percentage distribution (grey line, window size: 100) of the pHW-NS1(1-73)Dmd-GFP-NEP plasmid as determined by Illumina MiSeq sequencing after Covaris shearing and CLC Genomics Workbench version 7.0.3 data processing 12 . The diagram below the graph shows the organization of the different features from position 1 to position 4561 in the linearized pHW-NS1(1-73)Dmd-GFP-NEP plasmid. HCMV: human cytomegalovirus promoter, T: terminator sequence, NCR: non-coding region, NS1: non-structural protein 1, HA: hemagglutinin-tag, Dmd: dimerization domain (Dmd) of the Drosophila melanogaster Ncd protein, 2A FMDV: foot-and-mouth disease virus-2A auto processing site, 2A PTV-1: porcine teschovirus-1 2A cleavage site, NEP: nuclear export protein, Pol I: human RNA polymerase I promoter, polyA: polyA terminator, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene.
    Figure Legend Snippet: Sequence coverage of pHW-NS1(1-73)Dmd-GFP-NEP based on Illumina MiSeq sequencing. ( a ) Map of pHW-NS1(1-73)Dmd-GFP-NEP. ( b ) Sequence coverage (black line) and GC-percentage distribution (grey line, window size: 100) of the pHW-NS1(1-73)Dmd-GFP-NEP plasmid as determined by Illumina MiSeq sequencing after Covaris shearing and CLC Genomics Workbench version 7.0.3 data processing 12 . The diagram below the graph shows the organization of the different features from position 1 to position 4561 in the linearized pHW-NS1(1-73)Dmd-GFP-NEP plasmid. HCMV: human cytomegalovirus promoter, T: terminator sequence, NCR: non-coding region, NS1: non-structural protein 1, HA: hemagglutinin-tag, Dmd: dimerization domain (Dmd) of the Drosophila melanogaster Ncd protein, 2A FMDV: foot-and-mouth disease virus-2A auto processing site, 2A PTV-1: porcine teschovirus-1 2A cleavage site, NEP: nuclear export protein, Pol I: human RNA polymerase I promoter, polyA: polyA terminator, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene.

    Techniques Used: Sequencing, Gas Chromatography, Plasmid Preparation, Hemagglutination Assay

    Sequence coverage of the viral NS1(1-73)-GFP segment. Sequence coverage as determined by Illumina MiSeq sequencing after Nextera XT (orange) or Covaris (black) fragmentation and CLC Genomics Workbench version 7.0.3 data processing. The obtained sequences were filtered, trimmed and mapped to the reference sequence of the NS1(1-73)-GFP segment (based on the plasmid used to generate the recombinant PR8-NS1(1-73)GFP virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers) 12 . Below sequencing coverage plot: schematic representation of NS1(1-73)-GFP segment.
    Figure Legend Snippet: Sequence coverage of the viral NS1(1-73)-GFP segment. Sequence coverage as determined by Illumina MiSeq sequencing after Nextera XT (orange) or Covaris (black) fragmentation and CLC Genomics Workbench version 7.0.3 data processing. The obtained sequences were filtered, trimmed and mapped to the reference sequence of the NS1(1-73)-GFP segment (based on the plasmid used to generate the recombinant PR8-NS1(1-73)GFP virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers) 12 . Below sequencing coverage plot: schematic representation of NS1(1-73)-GFP segment.

    Techniques Used: Sequencing, Plasmid Preparation, Recombinant, Reverse Transcription Polymerase Chain Reaction

    Introducing silent mutations (C1398T and/or C1401T) that interrupt the ‘CCCGCC’ motif in the GFP coding sequence abrogates the drop in sequence coverage. The sequencing coverage is plotted in function of the nucleotide position in pHW-NS1(1-73)Dmd-GFP-NEP ( a ), pHW-NS1(1-73)Dmd-GFP-NEP C1398T ( b ), pHW-NS1(1-73)Dmd-GFP-NEP C1401T ( c ) or pHW-NS1(1-73)Dmd-GFP-NEP C1398T-C1401T ( d ) plasmid. Illumina MiSeq sequencing was performed after Covaris shearing and followed by CLC Genomics Workbench version 7.0.3 data processing and mapping of the reads to the plasmid reference sequence 12 . The position of the CCCGCC ( a ), CTCGCC ( b ), CCCGTC ( c ) and CTCGTC ( d ) motif is marked with an arrow head and the introduced mutation is marked in blue in this motif. The position of the sequence that codes for GFP is marked on the coverage plots in green.
    Figure Legend Snippet: Introducing silent mutations (C1398T and/or C1401T) that interrupt the ‘CCCGCC’ motif in the GFP coding sequence abrogates the drop in sequence coverage. The sequencing coverage is plotted in function of the nucleotide position in pHW-NS1(1-73)Dmd-GFP-NEP ( a ), pHW-NS1(1-73)Dmd-GFP-NEP C1398T ( b ), pHW-NS1(1-73)Dmd-GFP-NEP C1401T ( c ) or pHW-NS1(1-73)Dmd-GFP-NEP C1398T-C1401T ( d ) plasmid. Illumina MiSeq sequencing was performed after Covaris shearing and followed by CLC Genomics Workbench version 7.0.3 data processing and mapping of the reads to the plasmid reference sequence 12 . The position of the CCCGCC ( a ), CTCGCC ( b ), CCCGTC ( c ) and CTCGTC ( d ) motif is marked with an arrow head and the introduced mutation is marked in blue in this motif. The position of the sequence that codes for GFP is marked on the coverage plots in green.

    Techniques Used: Sequencing, Plasmid Preparation, Mutagenesis

    19) Product Images from "High-Resolution Microbial Community Succession of Microbially Induced Concrete Corrosion in Working Sanitary Manholes"

    Article Title: High-Resolution Microbial Community Succession of Microbially Induced Concrete Corrosion in Working Sanitary Manholes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116400

    Estimated bacterial community α-diversity in corrosion products as judged by Chao1 using Illumina MiSeq V1V2 16S amplicon sequencing (black square) and estimated pore water pH (grey diamond) after one to twelve months of exposure in a manhole environment. Error bars indicate 95% confidence intervals for 1,000 bootstrap calculations of Chao1.
    Figure Legend Snippet: Estimated bacterial community α-diversity in corrosion products as judged by Chao1 using Illumina MiSeq V1V2 16S amplicon sequencing (black square) and estimated pore water pH (grey diamond) after one to twelve months of exposure in a manhole environment. Error bars indicate 95% confidence intervals for 1,000 bootstrap calculations of Chao1.

    Techniques Used: Amplification, Sequencing

    Bacterial taxa observed in concrete surfaces and in corrosion products during three manhole monitoring campaigns by Illumina MiSeq of V1V2 rRNA amplicons. Taxa with greater than 1% representation in any sample are shown. Bar widths indicate relative percent abundance of microbial taxa in sample libraries; bar colors indicate taxa identity.
    Figure Legend Snippet: Bacterial taxa observed in concrete surfaces and in corrosion products during three manhole monitoring campaigns by Illumina MiSeq of V1V2 rRNA amplicons. Taxa with greater than 1% representation in any sample are shown. Bar widths indicate relative percent abundance of microbial taxa in sample libraries; bar colors indicate taxa identity.

    Techniques Used:

    20) Product Images from "Characterizing the Human Mycobiota: A Comparison of Small Subunit rRNA, ITS1, ITS2, and Large Subunit rRNA Genomic Targets"

    Article Title: Characterizing the Human Mycobiota: A Comparison of Small Subunit rRNA, ITS1, ITS2, and Large Subunit rRNA Genomic Targets

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02208

    Methodological overview. (A) The fungal ribosomal cistron, together with the primers and target regions assessed in this study. ITS, internal transcribed spacer; SSU, small subunit; LSU, large subunit. (B) Samples were collected from 21 fungal isolates, human upper-respiratory tract (sinonasal swab) ( n = 10), and mouse gut (fecal) material ( n = 10). All samples were amplified using primer pairs for four target genomic regions and sequenced on the Illumina MiSeq platform to compare the ability of each to accurately characterize the fungal communities in the samples. Readily available reference databases were also compared to assess the accuracy of taxonomic assignments of each of the sequence data sets.
    Figure Legend Snippet: Methodological overview. (A) The fungal ribosomal cistron, together with the primers and target regions assessed in this study. ITS, internal transcribed spacer; SSU, small subunit; LSU, large subunit. (B) Samples were collected from 21 fungal isolates, human upper-respiratory tract (sinonasal swab) ( n = 10), and mouse gut (fecal) material ( n = 10). All samples were amplified using primer pairs for four target genomic regions and sequenced on the Illumina MiSeq platform to compare the ability of each to accurately characterize the fungal communities in the samples. Readily available reference databases were also compared to assess the accuracy of taxonomic assignments of each of the sequence data sets.

    Techniques Used: Amplification, Sequencing

    Related Articles

    Diagnostic Assay:

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    Clone Assay:

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    Amplification:

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    Construct:

    Article Title: Molecular basis of wax-based color change and UV reflection in dragonflies
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    Article Title: Origin of wheat B-genome chromosomes inferred from RNA sequencing analysis of leaf transcripts from section Sitopsis species of Aegilops
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    Real-time Polymerase Chain Reaction:

    Article Title: Draft genome sequence and characterization of commensal Escherichia coli strain BG1 isolated from bovine gastro-intestinal tract
    Article Snippet: Library quality was assessed using the Agilent Bioanalyzer and libraries were quantified by qPCR using the Kapa Library Quantification Kit. .. DNA-seq experiments were performed on an Illumina MiSeq using a paired-end read length of 2 × 250 bp with the Illumina MiSeq Reagent Kits v2.

    Incubation:

    Article Title: Crenothrix are major methane consumers in stratified lakes
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    Article Title: Intense Exercise and Aerobic Conditioning Associated with Chromium or L-Carnitine Supplementation Modified the Fecal Microbiota of Fillies
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    Formalin-fixed Paraffin-Embedded:

    Article Title: Mutational and gene fusion analyses of primary large cell and large cell neuroendocrine lung cancer
    Article Snippet: Analysis of ALK , RET , and ROS1 gene fusions were performed using the RNA-based Archer FusionPlex ARR v2 kit (ArcherDX, Boulder, CO, US) and the MiSeq instrument (Illumina) ( ). .. The HCC78 (ROS1 -SLC34A2 ), KARPAS-299 (ALK -NPM1 ), LC-2/ad (CCDC6 -RET ), and H2228 (EML4 -ALK ) cell lines were used as controls ( ).

    Expressing:

    Article Title: Molecular basis of wax-based color change and UV reflection in dragonflies
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    Immunohistochemistry:

    Article Title: Mutational and gene fusion analyses of primary large cell and large cell neuroendocrine lung cancer
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    RAST Test:

    Article Title: ICESpy009, a Conjugative Genetic Element Carrying mef(E) in Streptococcus pyogenes
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    Genomic Sequencing:

    Article Title: Adenovirus Type 4 Respiratory Infections among Civilian Adults, Northeastern United States, 2011–2015
    Article Snippet: We prepared libraries with the NexteraXT kit (Illumina, San Diego, CA, USA) and performed paired-end sequencing on the Illumina MiSeq using the NextSeq 500 cycle v2 kit (Illumina). .. We prepared libraries with the NexteraXT kit (Illumina, San Diego, CA, USA) and performed paired-end sequencing on the Illumina MiSeq using the NextSeq 500 cycle v2 kit (Illumina).

    Article Title: ICESpy009, a Conjugative Genetic Element Carrying mef(E) in Streptococcus pyogenes
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    Infection:

    Article Title: Deep Sequencing of Foot-and-Mouth Disease Virus Reveals RNA Sequences Involved in Genome Packaging
    Article Snippet: RNA was extracted from samples of clarified infected cell lysate or purified virus using TRIzol and prepared for next-generation sequencing as described previously ( ). .. Subsequent DNase treatment and cleanup was followed by second-strand synthesis before library preparation using Nextera XT reagents (Illumina) and sequencing on the MiSeq v2 (Illumina).

    Digital PCR:

    Article Title: The mutational landscape of accelerated- and blast-phase myeloproliferative neoplasms impacts patient outcomes
    Article Snippet: Sequence data were analyzed by the NextGENe (v.2.3.1, SoftGenetics) and MiSeq Reporter (v2.4.60, MSR, Illumina) software packages. .. Data files from each sample were uploaded into Bench Laboratory NGS v4.2 (Agilent Technologies, Santa Clara, CA) for subsequent filtering to prioritize for reporting those variants that passed all MSR quality criteria, including depth of coverage of at least 100× and a variant allele frequency (VAF) threshold of > 15%.

    Generated:

    Article Title: Adenovirus Type 4 Respiratory Infections among Civilian Adults, Northeastern United States, 2011–2015
    Article Snippet: We prepared libraries with the NexteraXT kit (Illumina, San Diego, CA, USA) and performed paired-end sequencing on the Illumina MiSeq using the NextSeq 500 cycle v2 kit (Illumina). .. We prepared libraries with the NexteraXT kit (Illumina, San Diego, CA, USA) and performed paired-end sequencing on the Illumina MiSeq using the NextSeq 500 cycle v2 kit (Illumina).

    DNA Sequencing:

    Article Title: Draft genome sequence and characterization of commensal Escherichia coli strain BG1 isolated from bovine gastro-intestinal tract
    Article Snippet: Library quality was assessed using the Agilent Bioanalyzer and libraries were quantified by qPCR using the Kapa Library Quantification Kit. .. DNA-seq experiments were performed on an Illumina MiSeq using a paired-end read length of 2 × 250 bp with the Illumina MiSeq Reagent Kits v2. .. The raw reads were stored in ng6 [ ] and quality was checked using fastqc [ ].

    Polymerase Chain Reaction:

    Article Title: Genetic diversity and structure in Arapaima gigas populations from Amazon and Araguaia-Tocantins river basins
    Article Snippet: The library was then re-purified, to ensure no carry over of PCR primer dimer or other low size range amplicons ( < 200 bp) using an equal volume of AMPure magnetic beads (Perkin-Elmer, UK) and eluted in 15 μL EB buffer. .. Finally, the library was sequenced at the Institute of Aquaculture (University of Stirling, Scotland) on a single run on the Illumina MiSeq platform (162 bp paired end reads, 300 cycle kit, v2 chemistry kit; Illumina, Cambridge, UK).

    Article Title: Adenovirus Type 4 Respiratory Infections among Civilian Adults, Northeastern United States, 2011–2015
    Article Snippet: We prepared libraries with the NexteraXT kit (Illumina, San Diego, CA, USA) and performed paired-end sequencing on the Illumina MiSeq using the NextSeq 500 cycle v2 kit (Illumina). .. We assembled all genomic sequences de novo using the Illumina BaseSpace cloud application for SPAdes 3.5 ( ); we then remapped the sample sequences to the consensus sequence using Geneious Pro 9 ( ).

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    Article Title: Divergent short-chain fatty acid production and succession of colonic microbiota arise in fermentation of variously-sized wheat bran fractions
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    Article Title: Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania
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    Article Title: Natural soil microbes alter flowering phenology and the intensity of selection on flowering time in a wild Arabidopsis relative
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    Sonication:

    Article Title: Crenothrix are major methane consumers in stratified lakes
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    Article Title: Draft genome sequence and characterization of commensal Escherichia coli strain BG1 isolated from bovine gastro-intestinal tract
    Article Snippet: Briefly, DNA was fragmented by sonication using a Covaris M220 and adapters were ligated to be sequenced. .. DNA-seq experiments were performed on an Illumina MiSeq using a paired-end read length of 2 × 250 bp with the Illumina MiSeq Reagent Kits v2.

    DNA Extraction:

    Article Title: Subchronic low-dose 2,4-D exposure changed plasma acylcarnitine levels and induced gut microbiome perturbations in mice
    Article Snippet: Briefly, microbial DNA was extracted from mouse fecal pellets using the PowerSoil® DNA isolation kit (MoBio Laboratories, CA) according to manufacturer’s instructions. .. The quantification of DNA was performed using the Qubit 2.0 Fluorometer (Invitrogen, Life Technologies), and the DNA was sequenced using the Illumina MiSeq (500 cycles v2 kit) in the Georgia Genomics Facility of University of Georgia.

    Article Title: Crenothrix are major methane consumers in stratified lakes
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    Article Title: Intense Exercise and Aerobic Conditioning Associated with Chromium or L-Carnitine Supplementation Modified the Fecal Microbiota of Fillies
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    Article Title: Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania
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    Article Title: Natural soil microbes alter flowering phenology and the intensity of selection on flowering time in a wild Arabidopsis relative
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    RNA Sequencing Assay:

    Article Title: Molecular basis of wax-based color change and UV reflection in dragonflies
    Article Snippet: Using 1 µg of total RNA per sample as template, cDNA libraries were constructed using TruSeq RNA Sample Preparation Kits v2 (Illumina) and sequenced by HiSeq2000, Hiseq2500, or MiSeq (Illumina). .. Using 1 µg of total RNA per sample as template, cDNA libraries were constructed using TruSeq RNA Sample Preparation Kits v2 (Illumina) and sequenced by HiSeq2000, Hiseq2500, or MiSeq (Illumina).

    Article Title: Origin of wheat B-genome chromosomes inferred from RNA sequencing analysis of leaf transcripts from section Sitopsis species of Aegilops
    Article Snippet: Total RNA was extracted using Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan) from leaves of 2- to 3-month-old plants grown in a glass house. .. The extracted RNA was treated with DNase I at 37 °C for 20 min, after which paired-end libraries for RNA sequencing were constructed from 6 to 10 µg of total RNA using a TruSeq RNA Library Preparation kit v2 (Illumina, San Diego, CA, USA) according to a previously reported protocol and then sequenced with 300-bp paired-end reads on an Illumina MiSeq sequencer. .. The obtained reads were deposited in the DDBJ Sequence Read Archive under accession number DRA007097.

    Magnetic Beads:

    Article Title: Genetic diversity and structure in Arapaima gigas populations from Amazon and Araguaia-Tocantins river basins
    Article Snippet: The library was then re-purified, to ensure no carry over of PCR primer dimer or other low size range amplicons ( < 200 bp) using an equal volume of AMPure magnetic beads (Perkin-Elmer, UK) and eluted in 15 μL EB buffer. .. Finally, the library was sequenced at the Institute of Aquaculture (University of Stirling, Scotland) on a single run on the Illumina MiSeq platform (162 bp paired end reads, 300 cycle kit, v2 chemistry kit; Illumina, Cambridge, UK).

    Article Title: Intense Exercise and Aerobic Conditioning Associated with Chromium or L-Carnitine Supplementation Modified the Fecal Microbiota of Fillies
    Article Snippet: Amplicon libraries were purified with magnetic beads (Agencourt AMPure XP, Beckman Coulter Inc., Mississauga, ON) by mixing 72 μL of magnetic beads with 20 μL of the amplified libraries, and, after incubation at room temperature for 5 min, samples were washed with 80% ethanol twice and eluted in 40 μL of nuclease-free water. .. Sequencing was performed at the University of Guelph’s Advanced Analysis Centre, using an Illumina MiSeq platform (Illumina RTA v1.17.28; MCS v2.2) with the 2 x 250 V2 reagents.

    Isolation:

    Article Title: The mutational landscape of accelerated- and blast-phase myeloproliferative neoplasms impacts patient outcomes
    Article Snippet: Targeted sequencing was performed on DNA isolated from PB (n = 94) or bone marrow (n = 28) samples. .. Sequence data were analyzed by the NextGENe (v.2.3.1, SoftGenetics) and MiSeq Reporter (v2.4.60, MSR, Illumina) software packages.

    Purification:

    Article Title: Genetic diversity and structure in Arapaima gigas populations from Amazon and Araguaia-Tocantins river basins
    Article Snippet: The PCR reactions were combined (400 μL total) and column-purified with the MinElute PCR Purification Kit (Qiagen, UK), then eluted in 50 μL of EB buffer. .. Finally, the library was sequenced at the Institute of Aquaculture (University of Stirling, Scotland) on a single run on the Illumina MiSeq platform (162 bp paired end reads, 300 cycle kit, v2 chemistry kit; Illumina, Cambridge, UK).

    Article Title: Adenovirus Type 4 Respiratory Infections among Civilian Adults, Northeastern United States, 2011–2015
    Article Snippet: We then carried out next-generation sequencing reactions at the Wadsworth Center’s Applied Genomics Technology Core with the purified viral genomic DNA prepared at Lovelace Respiratory Research Institute. .. We prepared libraries with the NexteraXT kit (Illumina, San Diego, CA, USA) and performed paired-end sequencing on the Illumina MiSeq using the NextSeq 500 cycle v2 kit (Illumina).

    Article Title: Deep Sequencing of Foot-and-Mouth Disease Virus Reveals RNA Sequences Involved in Genome Packaging
    Article Snippet: RNA was extracted from samples of clarified infected cell lysate or purified virus using TRIzol and prepared for next-generation sequencing as described previously ( ). .. Subsequent DNase treatment and cleanup was followed by second-strand synthesis before library preparation using Nextera XT reagents (Illumina) and sequencing on the MiSeq v2 (Illumina).

    Article Title: Intense Exercise and Aerobic Conditioning Associated with Chromium or L-Carnitine Supplementation Modified the Fecal Microbiota of Fillies
    Article Snippet: Sequencing was performed at the University of Guelph’s Advanced Analysis Centre, using an Illumina MiSeq platform (Illumina RTA v1.17.28; MCS v2.2) with the 2 x 250 V2 reagents. .. Sequencing was performed at the University of Guelph’s Advanced Analysis Centre, using an Illumina MiSeq platform (Illumina RTA v1.17.28; MCS v2.2) with the 2 x 250 V2 reagents.

    Article Title: Divergent short-chain fatty acid production and succession of colonic microbiota arise in fermentation of variously-sized wheat bran fractions
    Article Snippet: PCR products were barcoded using the TruSeq dual-index approach, purified again using the AxyPrep PCR Clean-up Kit, quantitated via Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA), and pooled. .. Sequencing was performed using on an Illumina MiSeq run with 2 × 250 cycles and V2 chemistry (Illumina, Inc., San Diego, CA) at the Purdue Genomics Core Facility.

    Article Title: Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania
    Article Snippet: The purified libraries were then analyzed using an Agilent Bioanalyzer (Agilent, Santa Clara, CA, United States). .. Afterwards, the libraries were stored at -20°C until they were shipped on dry ice to California State University (Los Angeles, CA, United States) where they were sequenced on the Illumina Miseq with v2 300 cycle chemistry (Illumina, San Diego, CA, United States) to produce 250 bp paired-end reads.

    Article Title: Disease Severity and Immune Activity Relate to Distinct Interkingdom Gut Microbiome States in Ethnically Distinct Ulcerative Colitis Patients
    Article Snippet: ITS2 sequencing libraries were created with triplicate PCR amplicons per sample. .. Purified sequencing libraries were analyzed with a Bioanalyzer (Agilent), quantified with the Qubit HS dsDNA Assay kit (Invitrogen), and sequenced with an Illumina MiSeq platform and MiSeq Control Software v2.2.0 according to the manufacturer’s instructions (Illumina). .. FLASH v1.2.7, QIIME 1.8, and usearch software packages were used for sequence read quality filtering, operational taxonomic unit (OTU) picking, and OTU table generation ( ).

    Sequencing:

    Article Title: Subchronic low-dose 2,4-D exposure changed plasma acylcarnitine levels and induced gut microbiome perturbations in mice
    Article Snippet: Paragraph title: 16S rRNA gene sequencing and diversity analysis ... The quantification of DNA was performed using the Qubit 2.0 Fluorometer (Invitrogen, Life Technologies), and the DNA was sequenced using the Illumina MiSeq (500 cycles v2 kit) in the Georgia Genomics Facility of University of Georgia.

    Article Title: The Skin Microbiome of Cohabiting Couples
    Article Snippet: High-throughput sequencing on a MiSeq instrument (Illumina) was used to analyze the amplified V3-V4 region of the 16S rRNA gene. .. The MiSeq reagent v2 kit-500 cycles (Illumina) was used according to the manufacturer’s protocol to prepare the final quantified pool for sequencing. .. A 10% PhiX control was included to increase sample diversity.

    Article Title: Genetic diversity and structure in Arapaima gigas populations from Amazon and Araguaia-Tocantins river basins
    Article Snippet: Paragraph title: Library preparation and sequencing ... Finally, the library was sequenced at the Institute of Aquaculture (University of Stirling, Scotland) on a single run on the Illumina MiSeq platform (162 bp paired end reads, 300 cycle kit, v2 chemistry kit; Illumina, Cambridge, UK).

    Article Title: Molecular basis of wax-based color change and UV reflection in dragonflies
    Article Snippet: Using 1 µg of total RNA per sample as template, cDNA libraries were constructed using TruSeq RNA Sample Preparation Kits v2 (Illumina) and sequenced by HiSeq2000, Hiseq2500, or MiSeq (Illumina). .. Using 1 µg of total RNA per sample as template, cDNA libraries were constructed using TruSeq RNA Sample Preparation Kits v2 (Illumina) and sequenced by HiSeq2000, Hiseq2500, or MiSeq (Illumina).

    Article Title: Adenovirus Type 4 Respiratory Infections among Civilian Adults, Northeastern United States, 2011–2015
    Article Snippet: The 12 isolates that were selected for next-generation sequencing were representative of the set of identified variants on the basis of restriction enzyme analysis, location, and date of detection. .. We prepared libraries with the NexteraXT kit (Illumina, San Diego, CA, USA) and performed paired-end sequencing on the Illumina MiSeq using the NextSeq 500 cycle v2 kit (Illumina). .. We assembled all genomic sequences de novo using the Illumina BaseSpace cloud application for SPAdes 3.5 ( ); we then remapped the sample sequences to the consensus sequence using Geneious Pro 9 ( ).

    Article Title: Crenothrix are major methane consumers in stratified lakes
    Article Snippet: DNA from lake Zug was fragmented by sonication (MiSeq: 600–700 bp; HiSeq2500: 300 bp) using a Covaris S2 sonicator (Covaris, Woburn, MA, USA). .. The library was prepared using Ovation Ultra Low Library Systems V1 (for MiSeq) or V2 (for HiSeq2500) kits (NuGEN Technologies, San Carlos, CA, USA) and paired-end sequencing (2 × 300 or 2 × 150 bp) was performed using the Illumina MiSeq (2 × 300 bp) or HiSeq2500 (2 × 150 bp) platform (Illumina Inc., San Diego, CA, USA). .. DNA from Lake Rotsee was fragmented by sonication (350 bp) using a Covaris S2 sonicator (Covaris), the library was prepared using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) and paired-end sequencing (2 × 150 or 2 × 100 bp) was performed using the Illumina HiSeq2500 or 3000 platform (Illumina Inc.).

    Article Title: Draft genome sequence and characterization of commensal Escherichia coli strain BG1 isolated from bovine gastro-intestinal tract
    Article Snippet: Paragraph title: Genome sequencing and assembly ... DNA-seq experiments were performed on an Illumina MiSeq using a paired-end read length of 2 × 250 bp with the Illumina MiSeq Reagent Kits v2.

    Article Title: Deep Sequencing of Foot-and-Mouth Disease Virus Reveals RNA Sequences Involved in Genome Packaging
    Article Snippet: Briefly, reverse transcription used both random hexamers and FMDV-specific primers. .. Subsequent DNase treatment and cleanup was followed by second-strand synthesis before library preparation using Nextera XT reagents (Illumina) and sequencing on the MiSeq v2 (Illumina). .. Although originally described as a consensus-level sequencing methodology, depth of coverage was such that deep sequencing analysis also could be carried out.

    Article Title: The architecture of ArgR-DNA complexes at the genome-scale in Escherichia coli
    Article Snippet: Prior to the high-throughput sequencing, the sequencing libraries for ChIP-exo were cloned into TOPO vector (Invitrogen) and several colonies were subjected to Sanger sequencing to confirm the adapter sequences and inserted DNA length of the sequencing library. .. Then, the sequencing libraries were quantified using Qubit® 2.0 fluorometer (Invitrogen) and ExperionTM system (Bio-Rad), and sequenced using Illumina Miseq® V2 (Supplementary Figure S2). .. All sequencing reads from ChIP-exo experiments were mapped to E. coli MG1655 reference genome (NC_000913) using CLC Genomics Workbench5 with the length fraction of 0.9 and the similarity of 0.99 (Supplementary Table S1).

    Article Title: ICESpy009, a Conjugative Genetic Element Carrying mef(E) in Streptococcus pyogenes
    Article Snippet: Paragraph title: Sequencing and comparative analysis of ICE Spy 009. ... WGS of S. pyogenes MB56Spyo009 was carried out using Illumina MiSeq (PE 2 × 250 v2) technology (Illumina Inc., San Diego, CA).

    Article Title: The mutational landscape of accelerated- and blast-phase myeloproliferative neoplasms impacts patient outcomes
    Article Snippet: Analysis was performed using the TruSight Myeloid Sequencing Panel (Illumina, San Diego, CA) on the MiSeq platform (Illumina), as previously described., Fifty-four genes were profiled (39 genes tested for hotspot regions and 15 genes tested for complete exonic regions; supplemental Table 1A-B) using amplicon-based library preparation and (2 × 250 bp) paired-end sequencing using 50 ng input DNA. .. Sequence data were analyzed by the NextGENe (v.2.3.1, SoftGenetics) and MiSeq Reporter (v2.4.60, MSR, Illumina) software packages. .. Data files from each sample were uploaded into Bench Laboratory NGS v4.2 (Agilent Technologies, Santa Clara, CA) for subsequent filtering to prioritize for reporting those variants that passed all MSR quality criteria, including depth of coverage of at least 100× and a variant allele frequency (VAF) threshold of > 15%.

    Article Title: Intense Exercise and Aerobic Conditioning Associated with Chromium or L-Carnitine Supplementation Modified the Fecal Microbiota of Fillies
    Article Snippet: The purified samples were quantified in a spectrophotometer (NanoDrop, Thermo Fisher Scientific) and qualified on a 1% agarose gel. .. Sequencing was performed at the University of Guelph’s Advanced Analysis Centre, using an Illumina MiSeq platform (Illumina RTA v1.17.28; MCS v2.2) with the 2 x 250 V2 reagents. .. These data are available in the NCBI Sequence Read Archive under accession number PRJNA308944.

    Article Title: Immunogenomic analyses associate immunological alterations with mismatch repair defects in prostate cancer
    Article Snippet: Libraries were constructed from 40 ng DNA using GeneRead Mix-n-Match V2 (QIAGEN) customized 113 genes panel, and pooled libraries were sequenced on the MiSeq (Illumina). .. FASTQ files were generated using Illumina MiSeq Reporter v2.5.1.3.

    Article Title: Divergent short-chain fatty acid production and succession of colonic microbiota arise in fermentation of variously-sized wheat bran fractions
    Article Snippet: Quality control for pools was performed by running 1 µL of each pool on an Agilent Bioanalyzer with a High Sensitivity Chip (Agilent, Santa Clara, CA) followed by quantitation for pool loading via the KAPA Library Quantification Kit for Illumina Platforms. .. Sequencing was performed using on an Illumina MiSeq run with 2 × 250 cycles and V2 chemistry (Illumina, Inc., San Diego, CA) at the Purdue Genomics Core Facility. .. Sequences were processed using mothur v.1.39.3 according to the MiSeq SOP ( https://www.mothur.org/wiki/MiSeq_SOP [accessed 9/6/2017]) , with the following modifications.

    Article Title: Natural soil microbes alter flowering phenology and the intensity of selection on flowering time in a wild Arabidopsis relative
    Article Snippet: We extracted DNA from field-collected soil vouchers using the MoBioTM PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) and amplified variable region 4 of the bacterial 16S rRNA gene using established primer pairs 515F and 806R and PNA PCR clamps to reduce plastid and mitochondrial contamination ( ). .. Paired-end 2×250bp sequencing of barcoded amplicons was performed on a MiSeq machine running v2 chemistry (Illumina Inc., San Diego, CA, USA) at the Joint Genome Institute (Walnut Creek, CA, USA). .. The primer sequences were trimmed from the paired-end sequences, which were then overlapped and merged using FLASH ( ).

    Article Title: Origin of wheat B-genome chromosomes inferred from RNA sequencing analysis of leaf transcripts from section Sitopsis species of Aegilops
    Article Snippet: The extracted RNA was treated with DNase I at 37 °C for 20 min, after which paired-end libraries for RNA sequencing were constructed from 6 to 10 µg of total RNA using a TruSeq RNA Library Preparation kit v2 (Illumina, San Diego, CA, USA) according to a previously reported protocol and then sequenced with 300-bp paired-end reads on an Illumina MiSeq sequencer. .. The obtained reads were deposited in the DDBJ Sequence Read Archive under accession number DRA007097.

    Article Title: Disease Severity and Immune Activity Relate to Distinct Interkingdom Gut Microbiome States in Ethnically Distinct Ulcerative Colitis Patients
    Article Snippet: ITS2 sequencing libraries were created with triplicate PCR amplicons per sample. .. Purified sequencing libraries were analyzed with a Bioanalyzer (Agilent), quantified with the Qubit HS dsDNA Assay kit (Invitrogen), and sequenced with an Illumina MiSeq platform and MiSeq Control Software v2.2.0 according to the manufacturer’s instructions (Illumina). .. FLASH v1.2.7, QIIME 1.8, and usearch software packages were used for sequence read quality filtering, operational taxonomic unit (OTU) picking, and OTU table generation ( ).

    Agarose Gel Electrophoresis:

    Article Title: Genetic diversity and structure in Arapaima gigas populations from Amazon and Araguaia-Tocantins river basins
    Article Snippet: The purified pooled sample was separated by agarose gel (1.1%) electrophoresis and fragments ranging from approximately 450 to 650 bp were excised and purified using a MinElute Gel Extraction Kit (Qiagen, UK). .. Finally, the library was sequenced at the Institute of Aquaculture (University of Stirling, Scotland) on a single run on the Illumina MiSeq platform (162 bp paired end reads, 300 cycle kit, v2 chemistry kit; Illumina, Cambridge, UK).

    Article Title: Intense Exercise and Aerobic Conditioning Associated with Chromium or L-Carnitine Supplementation Modified the Fecal Microbiota of Fillies
    Article Snippet: Sequencing was performed at the University of Guelph’s Advanced Analysis Centre, using an Illumina MiSeq platform (Illumina RTA v1.17.28; MCS v2.2) with the 2 x 250 V2 reagents. .. Sequencing was performed at the University of Guelph’s Advanced Analysis Centre, using an Illumina MiSeq platform (Illumina RTA v1.17.28; MCS v2.2) with the 2 x 250 V2 reagents.

    Article Title: Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania
    Article Snippet: PCR products were pooled in equimolar ratios and were gel purified using a 2% agarose gel and a QIAquick Gel Extraction Kit per the manufacturer’s protocols (QIAGEN, Germantown, MD, United States). .. Afterwards, the libraries were stored at -20°C until they were shipped on dry ice to California State University (Los Angeles, CA, United States) where they were sequenced on the Illumina Miseq with v2 300 cycle chemistry (Illumina, San Diego, CA, United States) to produce 250 bp paired-end reads.

    Chromatin Immunoprecipitation:

    Article Title: The architecture of ArgR-DNA complexes at the genome-scale in Escherichia coli
    Article Snippet: Prior to the high-throughput sequencing, the sequencing libraries for ChIP-exo were cloned into TOPO vector (Invitrogen) and several colonies were subjected to Sanger sequencing to confirm the adapter sequences and inserted DNA length of the sequencing library. .. Then, the sequencing libraries were quantified using Qubit® 2.0 fluorometer (Invitrogen) and ExperionTM system (Bio-Rad), and sequenced using Illumina Miseq® V2 (Supplementary Figure S2).

    Article Title: Divergent short-chain fatty acid production and succession of colonic microbiota arise in fermentation of variously-sized wheat bran fractions
    Article Snippet: Quality control for pools was performed by running 1 µL of each pool on an Agilent Bioanalyzer with a High Sensitivity Chip (Agilent, Santa Clara, CA) followed by quantitation for pool loading via the KAPA Library Quantification Kit for Illumina Platforms. .. Sequencing was performed using on an Illumina MiSeq run with 2 × 250 cycles and V2 chemistry (Illumina, Inc., San Diego, CA) at the Purdue Genomics Core Facility.

    Plasmid Preparation:

    Article Title: The architecture of ArgR-DNA complexes at the genome-scale in Escherichia coli
    Article Snippet: Prior to the high-throughput sequencing, the sequencing libraries for ChIP-exo were cloned into TOPO vector (Invitrogen) and several colonies were subjected to Sanger sequencing to confirm the adapter sequences and inserted DNA length of the sequencing library. .. Then, the sequencing libraries were quantified using Qubit® 2.0 fluorometer (Invitrogen) and ExperionTM system (Bio-Rad), and sequenced using Illumina Miseq® V2 (Supplementary Figure S2).

    Software:

    Article Title: Mutational and gene fusion analyses of primary large cell and large cell neuroendocrine lung cancer
    Article Snippet: Analysis of ALK , RET , and ROS1 gene fusions were performed using the RNA-based Archer FusionPlex ARR v2 kit (ArcherDX, Boulder, CO, US) and the MiSeq instrument (Illumina) ( ). .. RNA from FFPE tissues (n = 11 samples) were extracted using the Qiagen Allprep FFPE extraction kit (Qiagen), while RNA from cell lines or fresh frozen tissue were extracted using the non-FFPE Qiagen Allprep extraction kit.

    Article Title: The mutational landscape of accelerated- and blast-phase myeloproliferative neoplasms impacts patient outcomes
    Article Snippet: Analysis was performed using the TruSight Myeloid Sequencing Panel (Illumina, San Diego, CA) on the MiSeq platform (Illumina), as previously described., Fifty-four genes were profiled (39 genes tested for hotspot regions and 15 genes tested for complete exonic regions; supplemental Table 1A-B) using amplicon-based library preparation and (2 × 250 bp) paired-end sequencing using 50 ng input DNA. .. Sequence data were analyzed by the NextGENe (v.2.3.1, SoftGenetics) and MiSeq Reporter (v2.4.60, MSR, Illumina) software packages. .. Data files from each sample were uploaded into Bench Laboratory NGS v4.2 (Agilent Technologies, Santa Clara, CA) for subsequent filtering to prioritize for reporting those variants that passed all MSR quality criteria, including depth of coverage of at least 100× and a variant allele frequency (VAF) threshold of > 15%.

    Article Title: Disease Severity and Immune Activity Relate to Distinct Interkingdom Gut Microbiome States in Ethnically Distinct Ulcerative Colitis Patients
    Article Snippet: ITS2 sequencing libraries were created with triplicate PCR amplicons per sample. .. Purified sequencing libraries were analyzed with a Bioanalyzer (Agilent), quantified with the Qubit HS dsDNA Assay kit (Invitrogen), and sequenced with an Illumina MiSeq platform and MiSeq Control Software v2.2.0 according to the manufacturer’s instructions (Illumina). .. FLASH v1.2.7, QIIME 1.8, and usearch software packages were used for sequence read quality filtering, operational taxonomic unit (OTU) picking, and OTU table generation ( ).

    Electrophoresis:

    Article Title: Genetic diversity and structure in Arapaima gigas populations from Amazon and Araguaia-Tocantins river basins
    Article Snippet: The purified pooled sample was separated by agarose gel (1.1%) electrophoresis and fragments ranging from approximately 450 to 650 bp were excised and purified using a MinElute Gel Extraction Kit (Qiagen, UK). .. Finally, the library was sequenced at the Institute of Aquaculture (University of Stirling, Scotland) on a single run on the Illumina MiSeq platform (162 bp paired end reads, 300 cycle kit, v2 chemistry kit; Illumina, Cambridge, UK).

    Multiplex Assay:

    Article Title: Multiplex PCR−Based Next-Generation Sequencing and Global Diversity of Seoul Virus in Humans and Rats
    Article Snippet: Paragraph title: Multiplex PCR–Based NGS ... We sequenced libraries by using the MiSeq benchtop sequencer (Illumina) with 2 × 150 bp and a MiSeq reagent V2 (Illumina).

    Sample Prep:

    Article Title: Molecular basis of wax-based color change and UV reflection in dragonflies
    Article Snippet: RNA sequencing was performed as described previously ( ). .. Using 1 µg of total RNA per sample as template, cDNA libraries were constructed using TruSeq RNA Sample Preparation Kits v2 (Illumina) and sequenced by HiSeq2000, Hiseq2500, or MiSeq (Illumina). .. The sequence data were deposited in the DNA Data Bank Japan Sequence Read Archive (accession numbers are shown in ).

    Article Title: Multiplex PCR−Based Next-Generation Sequencing and Global Diversity of Seoul Virus in Humans and Rats
    Article Snippet: Initial denaturation was at 95°C for 15 min, followed by 40 cycles or 25 cycles at 95°C for 20 s, 50°C for 40 s, and 72°C for 1 min, and a final elongation at 72°C for 3 min. We prepared multiplex PCR products by using the TruSeq Nano DNA LT Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. .. We sequenced libraries by using the MiSeq benchtop sequencer (Illumina) with 2 × 150 bp and a MiSeq reagent V2 (Illumina).

    In Situ:

    Article Title: Crenothrix are major methane consumers in stratified lakes
    Article Snippet: The following water volumes were filtered onto polycarbonate Nucleopore Tracked-Etched membrane filters (0.2 μm pore size; Whatman) and stored at −80 °C: 250 ml for in situ samples (R1 and Z1), 50 ml for Lake Rotsee incubations (R2 and R3) and 40 ml for Lake Zug incubations (Z2 and Z3). .. The library was prepared using Ovation Ultra Low Library Systems V1 (for MiSeq) or V2 (for HiSeq2500) kits (NuGEN Technologies, San Carlos, CA, USA) and paired-end sequencing (2 × 300 or 2 × 150 bp) was performed using the Illumina MiSeq (2 × 300 bp) or HiSeq2500 (2 × 150 bp) platform (Illumina Inc., San Diego, CA, USA).

    Next-Generation Sequencing:

    Article Title: Adenovirus Type 4 Respiratory Infections among Civilian Adults, Northeastern United States, 2011–2015
    Article Snippet: We prepared libraries with the NexteraXT kit (Illumina, San Diego, CA, USA) and performed paired-end sequencing on the Illumina MiSeq using the NextSeq 500 cycle v2 kit (Illumina). .. We prepared libraries with the NexteraXT kit (Illumina, San Diego, CA, USA) and performed paired-end sequencing on the Illumina MiSeq using the NextSeq 500 cycle v2 kit (Illumina).

    Article Title: Multiplex PCR−Based Next-Generation Sequencing and Global Diversity of Seoul Virus in Humans and Rats
    Article Snippet: Paragraph title: Multiplex PCR–Based NGS ... We sequenced libraries by using the MiSeq benchtop sequencer (Illumina) with 2 × 150 bp and a MiSeq reagent V2 (Illumina).

    Article Title: Deep Sequencing of Foot-and-Mouth Disease Virus Reveals RNA Sequences Involved in Genome Packaging
    Article Snippet: Paragraph title: Next-generation sequencing. ... Subsequent DNase treatment and cleanup was followed by second-strand synthesis before library preparation using Nextera XT reagents (Illumina) and sequencing on the MiSeq v2 (Illumina).

    Article Title: The architecture of ArgR-DNA complexes at the genome-scale in Escherichia coli
    Article Snippet: Paragraph title: Next-generation sequencing ... Then, the sequencing libraries were quantified using Qubit® 2.0 fluorometer (Invitrogen) and ExperionTM system (Bio-Rad), and sequenced using Illumina Miseq® V2 (Supplementary Figure S2).

    Article Title: Immunogenomic analyses associate immunological alterations with mismatch repair defects in prostate cancer
    Article Snippet: Paragraph title: Targeted NGS. ... Libraries were constructed from 40 ng DNA using GeneRead Mix-n-Match V2 (QIAGEN) customized 113 genes panel, and pooled libraries were sequenced on the MiSeq (Illumina).

    dsDNA Assay:

    Article Title: Disease Severity and Immune Activity Relate to Distinct Interkingdom Gut Microbiome States in Ethnically Distinct Ulcerative Colitis Patients
    Article Snippet: ITS2 sequencing libraries were created with triplicate PCR amplicons per sample. .. Purified sequencing libraries were analyzed with a Bioanalyzer (Agilent), quantified with the Qubit HS dsDNA Assay kit (Invitrogen), and sequenced with an Illumina MiSeq platform and MiSeq Control Software v2.2.0 according to the manufacturer’s instructions (Illumina). .. FLASH v1.2.7, QIIME 1.8, and usearch software packages were used for sequence read quality filtering, operational taxonomic unit (OTU) picking, and OTU table generation ( ).

    Quantitation Assay:

    Article Title: Divergent short-chain fatty acid production and succession of colonic microbiota arise in fermentation of variously-sized wheat bran fractions
    Article Snippet: Quality control for pools was performed by running 1 µL of each pool on an Agilent Bioanalyzer with a High Sensitivity Chip (Agilent, Santa Clara, CA) followed by quantitation for pool loading via the KAPA Library Quantification Kit for Illumina Platforms. .. Sequencing was performed using on an Illumina MiSeq run with 2 × 250 cycles and V2 chemistry (Illumina, Inc., San Diego, CA) at the Purdue Genomics Core Facility.

    Spectrophotometry:

    Article Title: Intense Exercise and Aerobic Conditioning Associated with Chromium or L-Carnitine Supplementation Modified the Fecal Microbiota of Fillies
    Article Snippet: Sequencing was performed at the University of Guelph’s Advanced Analysis Centre, using an Illumina MiSeq platform (Illumina RTA v1.17.28; MCS v2.2) with the 2 x 250 V2 reagents. .. Sequencing was performed at the University of Guelph’s Advanced Analysis Centre, using an Illumina MiSeq platform (Illumina RTA v1.17.28; MCS v2.2) with the 2 x 250 V2 reagents.

    Produced:

    Article Title: ICESpy009, a Conjugative Genetic Element Carrying mef(E) in Streptococcus pyogenes
    Article Snippet: WGS of S. pyogenes MB56Spyo009 was carried out using Illumina MiSeq (PE 2 × 250 v2) technology (Illumina Inc., San Diego, CA). .. WGS of S. pyogenes MB56Spyo009 was carried out using Illumina MiSeq (PE 2 × 250 v2) technology (Illumina Inc., San Diego, CA).

    High Throughput Screening Assay:

    Article Title: The Skin Microbiome of Cohabiting Couples
    Article Snippet: High-throughput sequencing on a MiSeq instrument (Illumina) was used to analyze the amplified V3-V4 region of the 16S rRNA gene. .. The MiSeq reagent v2 kit-500 cycles (Illumina) was used according to the manufacturer’s protocol to prepare the final quantified pool for sequencing.

    Article Title: The architecture of ArgR-DNA complexes at the genome-scale in Escherichia coli
    Article Snippet: Prior to the high-throughput sequencing, the sequencing libraries for ChIP-exo were cloned into TOPO vector (Invitrogen) and several colonies were subjected to Sanger sequencing to confirm the adapter sequences and inserted DNA length of the sequencing library. .. Then, the sequencing libraries were quantified using Qubit® 2.0 fluorometer (Invitrogen) and ExperionTM system (Bio-Rad), and sequenced using Illumina Miseq® V2 (Supplementary Figure S2).

    Gel Extraction:

    Article Title: Genetic diversity and structure in Arapaima gigas populations from Amazon and Araguaia-Tocantins river basins
    Article Snippet: The purified pooled sample was separated by agarose gel (1.1%) electrophoresis and fragments ranging from approximately 450 to 650 bp were excised and purified using a MinElute Gel Extraction Kit (Qiagen, UK). .. Finally, the library was sequenced at the Institute of Aquaculture (University of Stirling, Scotland) on a single run on the Illumina MiSeq platform (162 bp paired end reads, 300 cycle kit, v2 chemistry kit; Illumina, Cambridge, UK).

    Article Title: Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania
    Article Snippet: PCR products were pooled in equimolar ratios and were gel purified using a 2% agarose gel and a QIAquick Gel Extraction Kit per the manufacturer’s protocols (QIAGEN, Germantown, MD, United States). .. Afterwards, the libraries were stored at -20°C until they were shipped on dry ice to California State University (Los Angeles, CA, United States) where they were sequenced on the Illumina Miseq with v2 300 cycle chemistry (Illumina, San Diego, CA, United States) to produce 250 bp paired-end reads.

    Variant Assay:

    Article Title: Immunogenomic analyses associate immunological alterations with mismatch repair defects in prostate cancer
    Article Snippet: Libraries were constructed from 40 ng DNA using GeneRead Mix-n-Match V2 (QIAGEN) customized 113 genes panel, and pooled libraries were sequenced on the MiSeq (Illumina). .. FASTQ files were generated using Illumina MiSeq Reporter v2.5.1.3.

    Fluorescence In Situ Hybridization:

    Article Title: Mutational and gene fusion analyses of primary large cell and large cell neuroendocrine lung cancer
    Article Snippet: Analysis of ALK , RET , and ROS1 gene fusions were performed using the RNA-based Archer FusionPlex ARR v2 kit (ArcherDX, Boulder, CO, US) and the MiSeq instrument (Illumina) ( ). .. Data analysis was performed using software tools provided with the Archer kit (ArcherDX).

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    Principal coordinates analysis (PCoA) of unweighted UniFrac distances comparing the β-diversity of mini-microbial communities of spiked water among DNA-, PMA-, RNA-based methods. The mini-microbial communities compositions were as follow: ( a ) all live cells of Bacillus amyloliquefaciens , Mycobacterium smegmatis , Escherichia coli and Yersinia enterocolitica ; ( b ) all dead cells of the aforementioned species; ( c ) dead cells of B. amyloliquefaciens and M. smegmatis , and live cells of E. coli and Y. enterocolitica ; ( d ) live cells of B. amyloliquefaciens and M. smegmatis , and dead cells of E. coli and Y. enterocolitica ; determined using DNA-, PMA-, and RNA-based 16S rRNA <t>MiSeq</t> <t>Illumina</t> sequencing.
    Miseq, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 12675 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Principal coordinates analysis (PCoA) of unweighted UniFrac distances comparing the β-diversity of mini-microbial communities of spiked water among DNA-, PMA-, RNA-based methods. The mini-microbial communities compositions were as follow: ( a ) all live cells of Bacillus amyloliquefaciens , Mycobacterium smegmatis , Escherichia coli and Yersinia enterocolitica ; ( b ) all dead cells of the aforementioned species; ( c ) dead cells of B. amyloliquefaciens and M. smegmatis , and live cells of E. coli and Y. enterocolitica ; ( d ) live cells of B. amyloliquefaciens and M. smegmatis , and dead cells of E. coli and Y. enterocolitica ; determined using DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing.

    Journal: Scientific Reports

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water

    doi: 10.1038/s41598-017-02516-3

    Figure Lengend Snippet: Principal coordinates analysis (PCoA) of unweighted UniFrac distances comparing the β-diversity of mini-microbial communities of spiked water among DNA-, PMA-, RNA-based methods. The mini-microbial communities compositions were as follow: ( a ) all live cells of Bacillus amyloliquefaciens , Mycobacterium smegmatis , Escherichia coli and Yersinia enterocolitica ; ( b ) all dead cells of the aforementioned species; ( c ) dead cells of B. amyloliquefaciens and M. smegmatis , and live cells of E. coli and Y. enterocolitica ; ( d ) live cells of B. amyloliquefaciens and M. smegmatis , and dead cells of E. coli and Y. enterocolitica ; determined using DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing.

    Article Snippet: 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).

    Techniques: Sequencing

    Average sequences in mini-microbial communities of spiked water consisting of ( a ) all live cells of Bacillus amyloliquefaciens , Mycobacterium smegmatis , Escherichia coli and Yersinia enterocolitica ; ( b ) all dead cells of the aforementioned species; ( c ) dead cells of B. amyloliquefaciens and M. smegmatis , and live cells of E. coli and Y. enterocolitica; ( d ) live cells of B. amyloliquefaciens and M. smegmatis , and dead cells of E. coli and Y. enterocolitica ; determined using DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing. * P

    Journal: Scientific Reports

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water

    doi: 10.1038/s41598-017-02516-3

    Figure Lengend Snippet: Average sequences in mini-microbial communities of spiked water consisting of ( a ) all live cells of Bacillus amyloliquefaciens , Mycobacterium smegmatis , Escherichia coli and Yersinia enterocolitica ; ( b ) all dead cells of the aforementioned species; ( c ) dead cells of B. amyloliquefaciens and M. smegmatis , and live cells of E. coli and Y. enterocolitica; ( d ) live cells of B. amyloliquefaciens and M. smegmatis , and dead cells of E. coli and Y. enterocolitica ; determined using DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing. * P

    Article Snippet: 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).

    Techniques: Sequencing

    Average number of sequences in water samples collected from different sources analyzed using DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing. * P

    Journal: Scientific Reports

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water

    doi: 10.1038/s41598-017-02516-3

    Figure Lengend Snippet: Average number of sequences in water samples collected from different sources analyzed using DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing. * P

    Article Snippet: 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).

    Techniques: Sequencing

    Microbiota compositions in water samples collected from different sources determined using DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing.

    Journal: Scientific Reports

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water

    doi: 10.1038/s41598-017-02516-3

    Figure Lengend Snippet: Microbiota compositions in water samples collected from different sources determined using DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing.

    Article Snippet: 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).

    Techniques: Sequencing

    Compositions of mini-microbial communities of spiked water consisting of ( a ) all live cells of Bacillus amyloliquefaciens , Mycobacterium smegmatis , Escherichia coli and Yersinia enterocolitica ; ( b ) all dead cells of the aforementioned species; ( c ) dead cells of B. amyloliquefaciens and M. smegmatis , and live cells of E. coli and Y. enterocolitica; ( d ) live cells of B. amyloliquefaciens and M. smegmatis , and dead cells of E. coli and Y. enterocolitica ; determined using DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing.

    Journal: Scientific Reports

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water

    doi: 10.1038/s41598-017-02516-3

    Figure Lengend Snippet: Compositions of mini-microbial communities of spiked water consisting of ( a ) all live cells of Bacillus amyloliquefaciens , Mycobacterium smegmatis , Escherichia coli and Yersinia enterocolitica ; ( b ) all dead cells of the aforementioned species; ( c ) dead cells of B. amyloliquefaciens and M. smegmatis , and live cells of E. coli and Y. enterocolitica; ( d ) live cells of B. amyloliquefaciens and M. smegmatis , and dead cells of E. coli and Y. enterocolitica ; determined using DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing.

    Article Snippet: 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).

    Techniques: Sequencing

    α-diversity indices of microbiota of water samples collected from different sources and assessed by DNA-, PMA, and RNA-based 16S rRNA MiSeq Illumina sequencing.

    Journal: Scientific Reports

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water

    doi: 10.1038/s41598-017-02516-3

    Figure Lengend Snippet: α-diversity indices of microbiota of water samples collected from different sources and assessed by DNA-, PMA, and RNA-based 16S rRNA MiSeq Illumina sequencing.

    Article Snippet: 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).

    Techniques: Sequencing

    OTU compositions of mini-microbial communities in spiked water samples consisting of ( a ) all live cells of Bacillus amyloliquefaciens , Mycobacterium smegmatis , Escherichia coli and Yersinia enterocolitica ; ( b ) all dead cells of the aforementioned species; ( c ) dead cells of B. amyloliquefaciens and M. smegmatis , and live cells of E. coli and Y. enterocolitica; ( d ) live cells of B. amyloliquefaciens and M. smegmatis , and dead cells of E. coli and Y. enterocolitica ; determined by the DNA-, RNA-, and PMA-based 16S rRNA MiSeq Illumina sequencing.

    Journal: Scientific Reports

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water

    doi: 10.1038/s41598-017-02516-3

    Figure Lengend Snippet: OTU compositions of mini-microbial communities in spiked water samples consisting of ( a ) all live cells of Bacillus amyloliquefaciens , Mycobacterium smegmatis , Escherichia coli and Yersinia enterocolitica ; ( b ) all dead cells of the aforementioned species; ( c ) dead cells of B. amyloliquefaciens and M. smegmatis , and live cells of E. coli and Y. enterocolitica; ( d ) live cells of B. amyloliquefaciens and M. smegmatis , and dead cells of E. coli and Y. enterocolitica ; determined by the DNA-, RNA-, and PMA-based 16S rRNA MiSeq Illumina sequencing.

    Article Snippet: 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).

    Techniques: Sequencing

    Principal coordinates analysis (PCoA) of unweighted UniFrac distances of water samples collected from different sources to compare microbiota β-diversity between DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing.

    Journal: Scientific Reports

    Article Title: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water

    doi: 10.1038/s41598-017-02516-3

    Figure Lengend Snippet: Principal coordinates analysis (PCoA) of unweighted UniFrac distances of water samples collected from different sources to compare microbiota β-diversity between DNA-, PMA-, and RNA-based 16S rRNA MiSeq Illumina sequencing.

    Article Snippet: 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).

    Techniques: Sequencing

    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.

    Journal: BMC Genomics

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

    doi: 10.1186/s12864-015-1284-z

    Figure Lengend 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.

    Article Snippet: We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time.

    Techniques: 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.

    Journal: BMC Genomics

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

    doi: 10.1186/s12864-015-1284-z

    Figure Lengend 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.

    Article Snippet: We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time.

    Techniques: 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:

    Journal: BMC Genomics

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

    doi: 10.1186/s12864-015-1284-z

    Figure Lengend 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:

    Article Snippet: We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time.

    Techniques:

    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).

    Journal: BMC Genomics

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

    doi: 10.1186/s12864-015-1284-z

    Figure Lengend 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).

    Article Snippet: We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time.

    Techniques: 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).

    Journal: BMC Genomics

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

    doi: 10.1186/s12864-015-1284-z

    Figure Lengend 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).

    Article Snippet: We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time.

    Techniques: 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.

    Journal: BMC Genomics

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

    doi: 10.1186/s12864-015-1284-z

    Figure Lengend 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.

    Article Snippet: We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time.

    Techniques: Sequencing, Sample Prep, Ligation, Standard Deviation