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Illumina Inc illumina next generation sequencing
Statistical analysis of a de novo assembly of Cocos nucifera short reads generated by <t>Illumina</t> Hiseq™ 2000 sequencing. The distribution of assembled contigs and unigene lengths is shown (127,952 contigs and 57,304 unigenes were generated in the study).
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1) Product Images from "RNA-Seq Analysis of Cocos nucifera: Transcriptome Sequencing and De Novo Assembly for Subsequent Functional Genomics Approaches"

Article Title: RNA-Seq Analysis of Cocos nucifera: Transcriptome Sequencing and De Novo Assembly for Subsequent Functional Genomics Approaches

Journal: PLoS ONE

doi: 10.1371/journal.pone.0059997

Statistical analysis of a de novo assembly of Cocos nucifera short reads generated by Illumina Hiseq™ 2000 sequencing. The distribution of assembled contigs and unigene lengths is shown (127,952 contigs and 57,304 unigenes were generated in the study).
Figure Legend Snippet: Statistical analysis of a de novo assembly of Cocos nucifera short reads generated by Illumina Hiseq™ 2000 sequencing. The distribution of assembled contigs and unigene lengths is shown (127,952 contigs and 57,304 unigenes were generated in the study).

Techniques Used: Generated, Sequencing

2) Product Images from "SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing"

Article Title: SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-61779-228-1_9

RAD tag library generation. ( a ) Genomic DNA is digested with a restriction enzyme and a barcoded P1 adapter is ligated to the fragments. The P1 adapter contains a forward amplification primer site, an Illumina sequencing primer site, and a barcode ( shaded boxes represent P1 adapters with different barcodes). ( b ) Adapter-ligated fragments are combined (if multiplexing), sheared, and ( c ) ligated to a second adapter (P2, white boxes ). The P2 adapter is a divergent “Y” adapter, containing the reverse complement of the P2 reverse amplification primer site, preventing amplification of genomic fragments lacking a P1 adapter. ( d ).
Figure Legend Snippet: RAD tag library generation. ( a ) Genomic DNA is digested with a restriction enzyme and a barcoded P1 adapter is ligated to the fragments. The P1 adapter contains a forward amplification primer site, an Illumina sequencing primer site, and a barcode ( shaded boxes represent P1 adapters with different barcodes). ( b ) Adapter-ligated fragments are combined (if multiplexing), sheared, and ( c ) ligated to a second adapter (P2, white boxes ). The P2 adapter is a divergent “Y” adapter, containing the reverse complement of the P2 reverse amplification primer site, preventing amplification of genomic fragments lacking a P1 adapter. ( d ).

Techniques Used: Amplification, Sequencing, Multiplexing

Schematic diagram of population genomic data analysis using RAD sequencing. (a) Following Illumina sequencing of barcoded fragments, sequence reads ( thin lines ) are aligned to a reference genome sequence ( thick line ). Depth of coverage varies across tags. Reads that do not align to the genome, or align in multiple locations, are discarded. (b) Sample of reads at a single RAD site. The recognition site for the enzyme Sbf I is indicated along the reference genome sequence ( top ), and sequence reads typically proceed in both directions from this point, at which they overlap. At each nucleotide site, reads showing each of the four possible nucleotides can be tallied ( solid box ). (c) Nucleotide counts at each site for each individual are used in a maximum likelihood framework to assign the diploid genotype at the site. In this example, G/T heterozygote is the most likely genotype; the method provides the log-likelihood for this genotype, a maximum-likelihood estimate for the sequencing error rate ε, and a likelihood ratio test statistic comparing G/T to the second-most-likely genotype, G/G homozygote. (d) Each individual now has a diploid genotype at each nucleotide site sequenced, and single-nucleotide polymorphisms (SNPs) can be identified across populations. Note, however, that haplotype phase is still unknown across RAD tags. (e) SNPs ( ovals ) are distributed across the genome ( thick line ), and population genetic measures (e.g., F ST ) are calculated for each SNP. (f) A kernel smoothing average across multiple nucleotide positions is used to produce genome-wide distributions of population genetic measures.
Figure Legend Snippet: Schematic diagram of population genomic data analysis using RAD sequencing. (a) Following Illumina sequencing of barcoded fragments, sequence reads ( thin lines ) are aligned to a reference genome sequence ( thick line ). Depth of coverage varies across tags. Reads that do not align to the genome, or align in multiple locations, are discarded. (b) Sample of reads at a single RAD site. The recognition site for the enzyme Sbf I is indicated along the reference genome sequence ( top ), and sequence reads typically proceed in both directions from this point, at which they overlap. At each nucleotide site, reads showing each of the four possible nucleotides can be tallied ( solid box ). (c) Nucleotide counts at each site for each individual are used in a maximum likelihood framework to assign the diploid genotype at the site. In this example, G/T heterozygote is the most likely genotype; the method provides the log-likelihood for this genotype, a maximum-likelihood estimate for the sequencing error rate ε, and a likelihood ratio test statistic comparing G/T to the second-most-likely genotype, G/G homozygote. (d) Each individual now has a diploid genotype at each nucleotide site sequenced, and single-nucleotide polymorphisms (SNPs) can be identified across populations. Note, however, that haplotype phase is still unknown across RAD tags. (e) SNPs ( ovals ) are distributed across the genome ( thick line ), and population genetic measures (e.g., F ST ) are calculated for each SNP. (f) A kernel smoothing average across multiple nucleotide positions is used to produce genome-wide distributions of population genetic measures.

Techniques Used: Sequencing, Genome Wide

3) Product Images from "T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids"

Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids

Journal: Scientific Reports

doi: 10.1038/srep40767

Comparison of TOP-PCR to Illumina’s PCR method using serial dilutions of plasma cfDNA sample. Serial dilutions (5 ng–0.01 pg) of a plasma cfDNA sample isolated from a healthy female ( BBC ) was prepared and the cfDNA is amplified using either Illumina’s PCR or TOP-PCR. TOP panel: profiles generated by Illumina’s PCR method. Lower panel: profiles generated by TOP-PCR. Notice that the RFU values are no longer accurate because of figure overlay. PCR cycle numbers: 30 for 5–0.5 ng; 40 for 0.05 ng–0.01 pg. Size markers: 1 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
Figure Legend Snippet: Comparison of TOP-PCR to Illumina’s PCR method using serial dilutions of plasma cfDNA sample. Serial dilutions (5 ng–0.01 pg) of a plasma cfDNA sample isolated from a healthy female ( BBC ) was prepared and the cfDNA is amplified using either Illumina’s PCR or TOP-PCR. TOP panel: profiles generated by Illumina’s PCR method. Lower panel: profiles generated by TOP-PCR. Notice that the RFU values are no longer accurate because of figure overlay. PCR cycle numbers: 30 for 5–0.5 ng; 40 for 0.05 ng–0.01 pg. Size markers: 1 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

Techniques Used: Polymerase Chain Reaction, Isolation, Amplification, Generated

Comparison of TOP-PCR with Illumina’s PCR method using low amount of DNA as the input. ( a ) One micro-liter of original ovarian cancer plasma cfDNA sample with unknown concentration. ( b ) Same DNA sample but after 50 cycles of amplification by TOP-PCR. ( c ) Same DNA sample but after 50 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
Figure Legend Snippet: Comparison of TOP-PCR with Illumina’s PCR method using low amount of DNA as the input. ( a ) One micro-liter of original ovarian cancer plasma cfDNA sample with unknown concentration. ( b ) Same DNA sample but after 50 cycles of amplification by TOP-PCR. ( c ) Same DNA sample but after 50 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

Techniques Used: Polymerase Chain Reaction, Concentration Assay, Amplification

Comparison of TOP-PCT with Illumina’s PCR method using 20 ng of DNA. ( a ) One nano-gram original plasma cfDNA sample isolated from a healthy female (BBC). ( b ) Same DNA sample but after 20 cycles of amplification using TOP-PCR. ( c ) Same DNA sample but after 20 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
Figure Legend Snippet: Comparison of TOP-PCT with Illumina’s PCR method using 20 ng of DNA. ( a ) One nano-gram original plasma cfDNA sample isolated from a healthy female (BBC). ( b ) Same DNA sample but after 20 cycles of amplification using TOP-PCR. ( c ) Same DNA sample but after 20 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

Techniques Used: Polymerase Chain Reaction, Isolation, Amplification

4) Product Images from "Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus"

Article Title: Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus

Journal: Nature Communications

doi: 10.1038/ncomms13905

Paired nickase-induced lesions in the PAM-in orientation do not result in efficient strand separation and locus modification. ( a ) Schematic showing the position of gRNAs 8, 15, 11 and 32 on the HBB locus, alongside the predicted overhang length and PAM orientation (red). ( b ) Overall modification frequency resolved for deletions, insertions and gene conversion scored by Sanger sequencing of the amplified HBB locus in U2OS cells expressing the WT Cas9 variant and gRNA pairs 8/15 (PAM-out) or 11/32 (PAM-in). Data are represented as mean±s.e.m. Total number of sequences from four independent experiments is 324 for gRNA pair 8/15 and 280 for gRNA pair 11/32. ( c ) Overall modification frequency resolved for deletions, insertions and gene conversion scored by Sanger sequencing of the amplified HBB locus in U2OS cells expressing the N863A Cas9 variant and gRNA pair 8/15 (PAM-out) or the D10A Cas9 variant and gRNA pair 11/32 (PAM-in) in the presence (TREX2) or absence (CTRL) of the 3′–5′ exonuclease TREX2. gRNA expression was driven from PCR products containing the U6 promoter, gRNA sequence and TRACR (Methods). Data are represented as mean±s.e.m. The total number of sequences from three independent experiments is 203 for gRNA pair 8/15 CTRL, 572 for gRNA pair 11/32 CTRL, 224 for gRNA pair 8/15 TREX2 and 270 for gRNA pair 11/32 TREX2. ( d ) Box–whisker plot depicting the deletion size for either PAM-in gRNA pair 11/32 or PAM-out gRNA pair 8/15. Deletions were determined from sequences read by Illumina MiSeq. The line within the box depicts the median, with the box extending from the 25th to the 75th percentiles. The whiskers depict the minimum and maximum values within 1.5 inter-quartile range (IQR) of the median. Points with distance to median > 1.5 IQR are shown individually.
Figure Legend Snippet: Paired nickase-induced lesions in the PAM-in orientation do not result in efficient strand separation and locus modification. ( a ) Schematic showing the position of gRNAs 8, 15, 11 and 32 on the HBB locus, alongside the predicted overhang length and PAM orientation (red). ( b ) Overall modification frequency resolved for deletions, insertions and gene conversion scored by Sanger sequencing of the amplified HBB locus in U2OS cells expressing the WT Cas9 variant and gRNA pairs 8/15 (PAM-out) or 11/32 (PAM-in). Data are represented as mean±s.e.m. Total number of sequences from four independent experiments is 324 for gRNA pair 8/15 and 280 for gRNA pair 11/32. ( c ) Overall modification frequency resolved for deletions, insertions and gene conversion scored by Sanger sequencing of the amplified HBB locus in U2OS cells expressing the N863A Cas9 variant and gRNA pair 8/15 (PAM-out) or the D10A Cas9 variant and gRNA pair 11/32 (PAM-in) in the presence (TREX2) or absence (CTRL) of the 3′–5′ exonuclease TREX2. gRNA expression was driven from PCR products containing the U6 promoter, gRNA sequence and TRACR (Methods). Data are represented as mean±s.e.m. The total number of sequences from three independent experiments is 203 for gRNA pair 8/15 CTRL, 572 for gRNA pair 11/32 CTRL, 224 for gRNA pair 8/15 TREX2 and 270 for gRNA pair 11/32 TREX2. ( d ) Box–whisker plot depicting the deletion size for either PAM-in gRNA pair 11/32 or PAM-out gRNA pair 8/15. Deletions were determined from sequences read by Illumina MiSeq. The line within the box depicts the median, with the box extending from the 25th to the 75th percentiles. The whiskers depict the minimum and maximum values within 1.5 inter-quartile range (IQR) of the median. Points with distance to median > 1.5 IQR are shown individually.

Techniques Used: Modification, Sequencing, Amplification, Expressing, Variant Assay, Polymerase Chain Reaction, Whisker Assay

Characterization of repair pathway engagement of Cas9 variant-induced single nicks. ( a ) Frequency of deletions, insertions, gene conversion and gene correction (using the ‘high mismatch ssODN') from U2OS cells nucleofected with D10A Cas9 and N863A Cas9 and gRNA 8. Sequencing was performed using an Illumina MiSeq and modifications were quantified computationally (Methods). Three independent experiments. The P values for the difference in gene correction and gene conversion frequency was calculated using the two-tailed Student's t -test. Data are represented as mean±s.e.m. ( b ) Frequency of deletions, insertions, gene conversion and gene correction (using the ‘high mismatch ssODN') observed after U2OS cells were nucleofected with WT, D10A, or N863A Cas9 and gRNA 8, and an siRNA against RAD51 or firefly luciferase (FF) control. Sequencing was performed using an Illumina MiSeq and modifications were quantified computationally using (Methods). Three independent experiments. Data are represented as mean±s.e.m. ( c ) Bar graph of fold change in the rates of total editing events and gene correction (using the ‘high mismatch ssODN') for the D10A and N863A Cas9 mutants of cells treated with and siRNA against RAD51 relative to FF treated control. Three independent experiments. Data are represented as mean±s.e.m. ( d ) Representative histogram of the length of WT Cas9 and gRNA 8 or D10A Cas9 and gRNA 8-induced deletions in the presence or absence of RAD51.
Figure Legend Snippet: Characterization of repair pathway engagement of Cas9 variant-induced single nicks. ( a ) Frequency of deletions, insertions, gene conversion and gene correction (using the ‘high mismatch ssODN') from U2OS cells nucleofected with D10A Cas9 and N863A Cas9 and gRNA 8. Sequencing was performed using an Illumina MiSeq and modifications were quantified computationally (Methods). Three independent experiments. The P values for the difference in gene correction and gene conversion frequency was calculated using the two-tailed Student's t -test. Data are represented as mean±s.e.m. ( b ) Frequency of deletions, insertions, gene conversion and gene correction (using the ‘high mismatch ssODN') observed after U2OS cells were nucleofected with WT, D10A, or N863A Cas9 and gRNA 8, and an siRNA against RAD51 or firefly luciferase (FF) control. Sequencing was performed using an Illumina MiSeq and modifications were quantified computationally using (Methods). Three independent experiments. Data are represented as mean±s.e.m. ( c ) Bar graph of fold change in the rates of total editing events and gene correction (using the ‘high mismatch ssODN') for the D10A and N863A Cas9 mutants of cells treated with and siRNA against RAD51 relative to FF treated control. Three independent experiments. Data are represented as mean±s.e.m. ( d ) Representative histogram of the length of WT Cas9 and gRNA 8 or D10A Cas9 and gRNA 8-induced deletions in the presence or absence of RAD51.

Techniques Used: Variant Assay, Sequencing, Two Tailed Test, Luciferase

5) Product Images from "Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer"

Article Title: Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.25535

Comparison of miRNA expression levels obtained by NGS Illumina sequencing vs. TaqMan PCR. The results of the correlation are visualized as bar diagrams on the left and scatter diagrams with regression lines on the right comparing next generation sequencing (blue/y-axis) and TaqMan fold changes (red/x-axis) for hsa-miR-378a ( A ), hsa-miR-375 ( B ), hsa-miR-21-5p ( C ) and hsa-miR-215-5p ( D ). There was a good correlation of the NGS with the TaqMan results with a strong relationship for hsa-miR-378a (Pearson’s r = 0.9), hsa-miR-375 ( r = 0.77) and hsa-miR-215-5p ( r = 0.75), but only a moderate relationship for hsa-miR-21-5p ( r = 0.53).
Figure Legend Snippet: Comparison of miRNA expression levels obtained by NGS Illumina sequencing vs. TaqMan PCR. The results of the correlation are visualized as bar diagrams on the left and scatter diagrams with regression lines on the right comparing next generation sequencing (blue/y-axis) and TaqMan fold changes (red/x-axis) for hsa-miR-378a ( A ), hsa-miR-375 ( B ), hsa-miR-21-5p ( C ) and hsa-miR-215-5p ( D ). There was a good correlation of the NGS with the TaqMan results with a strong relationship for hsa-miR-378a (Pearson’s r = 0.9), hsa-miR-375 ( r = 0.77) and hsa-miR-215-5p ( r = 0.75), but only a moderate relationship for hsa-miR-21-5p ( r = 0.53).

Techniques Used: Expressing, Next-Generation Sequencing, Sequencing, Polymerase Chain Reaction

6) Product Images from "SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing"

Article Title: SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-61779-228-1_9

RAD tag library generation. ( a ) Genomic DNA is digested with a restriction enzyme and a barcoded P1 adapter is ligated to the fragments. The P1 adapter contains a forward amplification primer site, an Illumina sequencing primer site, and a barcode ( shaded boxes represent P1 adapters with different barcodes). ( b ) Adapter-ligated fragments are combined (if multiplexing), sheared, and ( c ) ligated to a second adapter (P2, white boxes ). The P2 adapter is a divergent “Y” adapter, containing the reverse complement of the P2 reverse amplification primer site, preventing amplification of genomic fragments lacking a P1 adapter. ( d ).
Figure Legend Snippet: RAD tag library generation. ( a ) Genomic DNA is digested with a restriction enzyme and a barcoded P1 adapter is ligated to the fragments. The P1 adapter contains a forward amplification primer site, an Illumina sequencing primer site, and a barcode ( shaded boxes represent P1 adapters with different barcodes). ( b ) Adapter-ligated fragments are combined (if multiplexing), sheared, and ( c ) ligated to a second adapter (P2, white boxes ). The P2 adapter is a divergent “Y” adapter, containing the reverse complement of the P2 reverse amplification primer site, preventing amplification of genomic fragments lacking a P1 adapter. ( d ).

Techniques Used: Amplification, Sequencing, Multiplexing

Schematic diagram of population genomic data analysis using RAD sequencing. (a) Following Illumina sequencing of barcoded fragments, sequence reads ( thin lines ) are aligned to a reference genome sequence ( thick line ). Depth of coverage varies across tags. Reads that do not align to the genome, or align in multiple locations, are discarded. (b) Sample of reads at a single RAD site. The recognition site for the enzyme Sbf I is indicated along the reference genome sequence ( top ), and sequence reads typically proceed in both directions from this point, at which they overlap. At each nucleotide site, reads showing each of the four possible nucleotides can be tallied ( solid box ). (c) Nucleotide counts at each site for each individual are used in a maximum likelihood framework to assign the diploid genotype at the site. In this example, G/T heterozygote is the most likely genotype; the method provides the log-likelihood for this genotype, a maximum-likelihood estimate for the sequencing error rate ε, and a likelihood ratio test statistic comparing G/T to the second-most-likely genotype, G/G homozygote. (d) Each individual now has a diploid genotype at each nucleotide site sequenced, and single-nucleotide polymorphisms (SNPs) can be identified across populations. Note, however, that haplotype phase is still unknown across RAD tags. (e) SNPs ( ovals ) are distributed across the genome ( thick line ), and population genetic measures (e.g., F ST ) are calculated for each SNP. (f) A kernel smoothing average across multiple nucleotide positions is used to produce genome-wide distributions of population genetic measures.
Figure Legend Snippet: Schematic diagram of population genomic data analysis using RAD sequencing. (a) Following Illumina sequencing of barcoded fragments, sequence reads ( thin lines ) are aligned to a reference genome sequence ( thick line ). Depth of coverage varies across tags. Reads that do not align to the genome, or align in multiple locations, are discarded. (b) Sample of reads at a single RAD site. The recognition site for the enzyme Sbf I is indicated along the reference genome sequence ( top ), and sequence reads typically proceed in both directions from this point, at which they overlap. At each nucleotide site, reads showing each of the four possible nucleotides can be tallied ( solid box ). (c) Nucleotide counts at each site for each individual are used in a maximum likelihood framework to assign the diploid genotype at the site. In this example, G/T heterozygote is the most likely genotype; the method provides the log-likelihood for this genotype, a maximum-likelihood estimate for the sequencing error rate ε, and a likelihood ratio test statistic comparing G/T to the second-most-likely genotype, G/G homozygote. (d) Each individual now has a diploid genotype at each nucleotide site sequenced, and single-nucleotide polymorphisms (SNPs) can be identified across populations. Note, however, that haplotype phase is still unknown across RAD tags. (e) SNPs ( ovals ) are distributed across the genome ( thick line ), and population genetic measures (e.g., F ST ) are calculated for each SNP. (f) A kernel smoothing average across multiple nucleotide positions is used to produce genome-wide distributions of population genetic measures.

Techniques Used: Sequencing, Genome Wide

7) Product Images from "Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis"

Article Title: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis

Journal: Cell Discovery

doi: 10.1038/celldisc.2017.31

Comparison of A. membranaceus transcriptomes acquired using different sequencing platforms. ( a ) Length distribution of Iso-Seq consensus transcripts compared with de novo assembled transcripts from Illumina sequencing platform. N50 length similar to the median contig or assembled sequence length and is defined as the length N for which 50% of all bases in the assembled sequences (either Illumina de novo assembled transcript or Iso-Seq consensus transcript) are in an assembled sequence of length L
Figure Legend Snippet: Comparison of A. membranaceus transcriptomes acquired using different sequencing platforms. ( a ) Length distribution of Iso-Seq consensus transcripts compared with de novo assembled transcripts from Illumina sequencing platform. N50 length similar to the median contig or assembled sequence length and is defined as the length N for which 50% of all bases in the assembled sequences (either Illumina de novo assembled transcript or Iso-Seq consensus transcript) are in an assembled sequence of length L

Techniques Used: Sequencing

8) Product Images from "Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer"

Article Title: Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.25535

Comparison of miRNA expression levels obtained by NGS Illumina sequencing vs. TaqMan PCR. The results of the correlation are visualized as bar diagrams on the left and scatter diagrams with regression lines on the right comparing next generation sequencing (blue/y-axis) and TaqMan fold changes (red/x-axis) for hsa-miR-378a ( A ), hsa-miR-375 ( B ), hsa-miR-21-5p ( C ) and hsa-miR-215-5p ( D ). There was a good correlation of the NGS with the TaqMan results with a strong relationship for hsa-miR-378a (Pearson’s r = 0.9), hsa-miR-375 ( r = 0.77) and hsa-miR-215-5p ( r = 0.75), but only a moderate relationship for hsa-miR-21-5p ( r = 0.53).
Figure Legend Snippet: Comparison of miRNA expression levels obtained by NGS Illumina sequencing vs. TaqMan PCR. The results of the correlation are visualized as bar diagrams on the left and scatter diagrams with regression lines on the right comparing next generation sequencing (blue/y-axis) and TaqMan fold changes (red/x-axis) for hsa-miR-378a ( A ), hsa-miR-375 ( B ), hsa-miR-21-5p ( C ) and hsa-miR-215-5p ( D ). There was a good correlation of the NGS with the TaqMan results with a strong relationship for hsa-miR-378a (Pearson’s r = 0.9), hsa-miR-375 ( r = 0.77) and hsa-miR-215-5p ( r = 0.75), but only a moderate relationship for hsa-miR-21-5p ( r = 0.53).

Techniques Used: Expressing, Next-Generation Sequencing, Sequencing, Polymerase Chain Reaction

9) Product Images from "YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs"

Article Title: YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx005

Adapter-tRNA ligation efficiencies with a conventional method and YAMAT-seq. ( A ) Schematic representation of the adapter ligation reactions used in the conventional and YAMAT-seq methods. In the conventional method, tRNA was subjected to 3΄-adapter (3΄-AD) and 5΄-adapter (5΄-AD) ligations catalyzed by truncated Rnl2 and Rnl1, respectively, using the Illumina TruSeq Small RNA Sample Preparation Kit. In contrast, in the YAMAT-seq method, a Y-shaped adapter (Y-AD) is ligated to a tRNA by Rnl2. ( B ) Ligation products of synthetic human cyto tRNA AspGUC and 5΄-AD or 3΄-AD were quantified by real-time qRT-PCR. The amounts of ligation products were determined based on standard curves, and ligation efficiencies were calculated as percentages of ligated tRNA versus input tRNA (set as 100%). Each data set represents the average of three independent experiments with bars showing the SD. ( C ) Ligation products of the indicated native cyto tRNAs in BT-474 total RNA and 5΄-AD or 3΄-AD were quantified by real-time qRT-PCR. Each data set represents the average Ct values of three independent experiments with bars showing the SD. ( D ) Image of 8% native polyacrylamide gel electrophoresis (PAGE) of amplified cDNAs resulting from BT-474 total RNA sequencing by conventional and YAMAT-seq procedures. The total adapter lengths of the conventional and YAMAT-seq procedures are 118 and 140 bp, respectively. The YAMAT-seq-derived cDNAs in the designated Bands 1–3 regions were gel-purified and subjected to cloning; the identified sequences are shown in Supplementary Table S1 . The cDNAs in regions designated with gray lines are expected to contain tRNA fraction.
Figure Legend Snippet: Adapter-tRNA ligation efficiencies with a conventional method and YAMAT-seq. ( A ) Schematic representation of the adapter ligation reactions used in the conventional and YAMAT-seq methods. In the conventional method, tRNA was subjected to 3΄-adapter (3΄-AD) and 5΄-adapter (5΄-AD) ligations catalyzed by truncated Rnl2 and Rnl1, respectively, using the Illumina TruSeq Small RNA Sample Preparation Kit. In contrast, in the YAMAT-seq method, a Y-shaped adapter (Y-AD) is ligated to a tRNA by Rnl2. ( B ) Ligation products of synthetic human cyto tRNA AspGUC and 5΄-AD or 3΄-AD were quantified by real-time qRT-PCR. The amounts of ligation products were determined based on standard curves, and ligation efficiencies were calculated as percentages of ligated tRNA versus input tRNA (set as 100%). Each data set represents the average of three independent experiments with bars showing the SD. ( C ) Ligation products of the indicated native cyto tRNAs in BT-474 total RNA and 5΄-AD or 3΄-AD were quantified by real-time qRT-PCR. Each data set represents the average Ct values of three independent experiments with bars showing the SD. ( D ) Image of 8% native polyacrylamide gel electrophoresis (PAGE) of amplified cDNAs resulting from BT-474 total RNA sequencing by conventional and YAMAT-seq procedures. The total adapter lengths of the conventional and YAMAT-seq procedures are 118 and 140 bp, respectively. The YAMAT-seq-derived cDNAs in the designated Bands 1–3 regions were gel-purified and subjected to cloning; the identified sequences are shown in Supplementary Table S1 . The cDNAs in regions designated with gray lines are expected to contain tRNA fraction.

Techniques Used: Ligation, Sample Prep, Quantitative RT-PCR, Polyacrylamide Gel Electrophoresis, Amplification, RNA Sequencing Assay, Derivative Assay, Purification, Clone Assay

Amplification and sequencing of mature tRNAs using YAMAT-seq. ( A ) Image of 8% native PAGE of amplified cDNAs resulting from YAMAT-seq of total RNAs from MCF-7, SK-BR-3 and BT-20 cells. The result of one of the triplicate, Replicate 1 (R1), from each cell line is shown. The region designated with a gray line was subjected to gel-purification and Illumina sequencing. ( B ) Flow diagram of the read numbers filtered by the indicated sequence analyses. The result of R1 from each cell line is shown, whereas the results of R2 and R3 are shown in Supplementary Figure S1 . ( C ) Spearman's rank correlation analysis using tRNA reads of the technical triplicates (R1–R3) from each cell line. The results using the reads of all tRNAs (left), cyto tRNAs (middle) and mt tRNAs (right) are shown. The color bar indicates the strength of Spearman's correlation coefficient.
Figure Legend Snippet: Amplification and sequencing of mature tRNAs using YAMAT-seq. ( A ) Image of 8% native PAGE of amplified cDNAs resulting from YAMAT-seq of total RNAs from MCF-7, SK-BR-3 and BT-20 cells. The result of one of the triplicate, Replicate 1 (R1), from each cell line is shown. The region designated with a gray line was subjected to gel-purification and Illumina sequencing. ( B ) Flow diagram of the read numbers filtered by the indicated sequence analyses. The result of R1 from each cell line is shown, whereas the results of R2 and R3 are shown in Supplementary Figure S1 . ( C ) Spearman's rank correlation analysis using tRNA reads of the technical triplicates (R1–R3) from each cell line. The results using the reads of all tRNAs (left), cyto tRNAs (middle) and mt tRNAs (right) are shown. The color bar indicates the strength of Spearman's correlation coefficient.

Techniques Used: Amplification, Sequencing, Clear Native PAGE, Gel Purification, Flow Cytometry

Schematic representation of mature tRNA sequencing by YAMAT-seq. Initially, amino acids at the 3΄-ends of mature aminoacylated tRNAs are removed by deacylation treatment. A DNA/RNA hybrid Y-shaped adapter is then specifically hybridized to these deacylated mature tRNAs. The bold line regions of the adapter contain identical sequences to those used for Illumina sequencing. Following hybridization, Rnl2 ligates nicks between the adapter and mature tRNA, and the resultant ligation product is amplified, gel-purified and subjected to Illumina sequencing.
Figure Legend Snippet: Schematic representation of mature tRNA sequencing by YAMAT-seq. Initially, amino acids at the 3΄-ends of mature aminoacylated tRNAs are removed by deacylation treatment. A DNA/RNA hybrid Y-shaped adapter is then specifically hybridized to these deacylated mature tRNAs. The bold line regions of the adapter contain identical sequences to those used for Illumina sequencing. Following hybridization, Rnl2 ligates nicks between the adapter and mature tRNA, and the resultant ligation product is amplified, gel-purified and subjected to Illumina sequencing.

Techniques Used: Sequencing, Hybridization, Ligation, Amplification, Purification

10) Product Images from "Methanogenic Degradation of Long n-Alkanes Requires Fumarate-Dependent Activation"

Article Title: Methanogenic Degradation of Long n-Alkanes Requires Fumarate-Dependent Activation

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00985-19

Microbial community composition recovered from Illumina sequencing of 16S rRNA genes in methanogenic enrichment cultures amended with C 16 to C 20 n -alkanes (C 16 -C 20 ) and the control cultures not amended with n -alkane (control). (A) Bacterial community composition. Only taxa abundant (at the family level) in more than 5% of the total bacterial reads in at least one sample are shown. (B) Archaeal community composition. Only taxa abundant (at the genus level) in more than 1% of the total archaeal reads in at least one sample are shown.
Figure Legend Snippet: Microbial community composition recovered from Illumina sequencing of 16S rRNA genes in methanogenic enrichment cultures amended with C 16 to C 20 n -alkanes (C 16 -C 20 ) and the control cultures not amended with n -alkane (control). (A) Bacterial community composition. Only taxa abundant (at the family level) in more than 5% of the total bacterial reads in at least one sample are shown. (B) Archaeal community composition. Only taxa abundant (at the genus level) in more than 1% of the total archaeal reads in at least one sample are shown.

Techniques Used: Sequencing

11) Product Images from "Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis"

Article Title: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis

Journal: Cell Discovery

doi: 10.1038/celldisc.2017.31

Comparison of A. membranaceus transcriptomes acquired using different sequencing platforms. ( a ) Length distribution of Iso-Seq consensus transcripts compared with de novo assembled transcripts from Illumina sequencing platform. N50 length similar to the median contig or assembled sequence length and is defined as the length N for which 50% of all bases in the assembled sequences (either Illumina de novo assembled transcript or Iso-Seq consensus transcript) are in an assembled sequence of length L
Figure Legend Snippet: Comparison of A. membranaceus transcriptomes acquired using different sequencing platforms. ( a ) Length distribution of Iso-Seq consensus transcripts compared with de novo assembled transcripts from Illumina sequencing platform. N50 length similar to the median contig or assembled sequence length and is defined as the length N for which 50% of all bases in the assembled sequences (either Illumina de novo assembled transcript or Iso-Seq consensus transcript) are in an assembled sequence of length L

Techniques Used: Sequencing

12) Product Images from "Optimized Illumina PCR-free library preparation for bacterial whole genome sequencing and analysis of factors influencing de novo assembly"

Article Title: Optimized Illumina PCR-free library preparation for bacterial whole genome sequencing and analysis of factors influencing de novo assembly

Journal: BMC Research Notes

doi: 10.1186/s13104-016-2072-9

Insert size distributions after second size selection. Data originate from analysis with Bioanalyzer instruments. a Insert size distributions of sequencing libraries obtained with the standard Illumina TruSeq ® DNA PCR-free LPP have an overall good reproducibility. All sequenced TS libraries are shown. b Modifications during DNA fragmentation and insert size selection enabled the creation of sequencing libraries with sharper and more symmetric insert size distributions. Sequencing libraries Efa_TS and Efa_IS1 are illustrated in red and blue , respectively. c In addition, different RB:BB ratios led to sequencing libraries varying in average insert size for the same genome. Blue : Pst_IS1, red : Pst_IS3. d Insert size distribution reproducibility is maintained when using modified LPPs
Figure Legend Snippet: Insert size distributions after second size selection. Data originate from analysis with Bioanalyzer instruments. a Insert size distributions of sequencing libraries obtained with the standard Illumina TruSeq ® DNA PCR-free LPP have an overall good reproducibility. All sequenced TS libraries are shown. b Modifications during DNA fragmentation and insert size selection enabled the creation of sequencing libraries with sharper and more symmetric insert size distributions. Sequencing libraries Efa_TS and Efa_IS1 are illustrated in red and blue , respectively. c In addition, different RB:BB ratios led to sequencing libraries varying in average insert size for the same genome. Blue : Pst_IS1, red : Pst_IS3. d Insert size distribution reproducibility is maintained when using modified LPPs

Techniques Used: Selection, Sequencing, Polymerase Chain Reaction, Modification

13) Product Images from "Influenza A/H3N2 virus infection in immunocompromised ferrets and emergence of antiviral resistance"

Article Title: Influenza A/H3N2 virus infection in immunocompromised ferrets and emergence of antiviral resistance

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200849

The frequency of the R292K mutation as determined by Illumina NGS in Oseltamivir treated immunocompromised ferrets. The percentage of the R292K variant was determined in pooled throat/nose samples collected at 2, 4, 6, 8 and 10 dpi from immunocompromised ferrets that received Oseltamivir treatment. The graph depicts the frequency of the R292K mutation over time for each ferret separately, with circles, squares, triangles, down triangles, diamonds and open circles representing ferrets 1–6 respectively.
Figure Legend Snippet: The frequency of the R292K mutation as determined by Illumina NGS in Oseltamivir treated immunocompromised ferrets. The percentage of the R292K variant was determined in pooled throat/nose samples collected at 2, 4, 6, 8 and 10 dpi from immunocompromised ferrets that received Oseltamivir treatment. The graph depicts the frequency of the R292K mutation over time for each ferret separately, with circles, squares, triangles, down triangles, diamonds and open circles representing ferrets 1–6 respectively.

Techniques Used: Mutagenesis, Next-Generation Sequencing, Variant Assay

14) Product Images from "The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms"

Article Title: The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01692-15

Ordination plots of bacterial community structures as determined by nonmetric multidimensional scaling (nMDS) of PCR-Illumina profiles of 16S rRNA gene fragments in the BAC filters at SPRWS. (A) Bacterial community dynamics in filter 3. (B) Bacterial community dynamics in filter 6. (C) Bacterial community dynamics in filter 13. (D) Comparison of bacterial community structures in different filters on 1 July 2011; different filters are represented by a number. ●, 15 March 2011; ■, 31 May 2011; ▲, 1 July 2011; ◆, 11 August 2011; ○, 22 November 2011; □, 15 December 2011; △, 14 February 2012. The data are shown as the arithmetic means of triplicate profiles (±1 standard deviation).
Figure Legend Snippet: Ordination plots of bacterial community structures as determined by nonmetric multidimensional scaling (nMDS) of PCR-Illumina profiles of 16S rRNA gene fragments in the BAC filters at SPRWS. (A) Bacterial community dynamics in filter 3. (B) Bacterial community dynamics in filter 6. (C) Bacterial community dynamics in filter 13. (D) Comparison of bacterial community structures in different filters on 1 July 2011; different filters are represented by a number. ●, 15 March 2011; ■, 31 May 2011; ▲, 1 July 2011; ◆, 11 August 2011; ○, 22 November 2011; □, 15 December 2011; △, 14 February 2012. The data are shown as the arithmetic means of triplicate profiles (±1 standard deviation).

Techniques Used: Polymerase Chain Reaction, BAC Assay, Standard Deviation

(A) Dynamics of several of the most dominant bacterial populations in filter 3 at SPRWS in the PCR-Illumina profiles. (B) Comparison of the representative, individual bacterial populations in different filters on 1 July 2011. The PCR-Illumina data for all bacterial profiles on all dates are included in the supplemental material.
Figure Legend Snippet: (A) Dynamics of several of the most dominant bacterial populations in filter 3 at SPRWS in the PCR-Illumina profiles. (B) Comparison of the representative, individual bacterial populations in different filters on 1 July 2011. The PCR-Illumina data for all bacterial profiles on all dates are included in the supplemental material.

Techniques Used: Polymerase Chain Reaction

Comparison of the relative quantities of ammonia-oxidizing and nitrite-oxidizing bacteria in three SPRWS BAC filters. The values for AOB (Illumina) and NOB (Illumina) represent the fractions of known AOB and Nitrospira sequences, respectively, found in an Illumina profile divided by the total number of sequences in that profile. The amoA /16S rRNA represents the ratio of amoA to 16S rRNA genes as measured by quantitative real-time PCR (see Data Sets S2 to S4 in the supplemental material).
Figure Legend Snippet: Comparison of the relative quantities of ammonia-oxidizing and nitrite-oxidizing bacteria in three SPRWS BAC filters. The values for AOB (Illumina) and NOB (Illumina) represent the fractions of known AOB and Nitrospira sequences, respectively, found in an Illumina profile divided by the total number of sequences in that profile. The amoA /16S rRNA represents the ratio of amoA to 16S rRNA genes as measured by quantitative real-time PCR (see Data Sets S2 to S4 in the supplemental material).

Techniques Used: BAC Assay, Real-time Polymerase Chain Reaction

15) Product Images from "De novo Assembly and Characterization of the Testis Transcriptome and Development of EST-SSR Markers in the Cockroach Periplaneta americana"

Article Title: De novo Assembly and Characterization of the Testis Transcriptome and Development of EST-SSR Markers in the Cockroach Periplaneta americana

Journal: Scientific Reports

doi: 10.1038/srep11144

Species distribution for homology search of Illumina sequences against the nr database. Species distribution is shown as percentage of the total number of homologous sequences with an E-value cutoff of 1.0E −5 . The most significant hit of each sequence was used for further analysis.
Figure Legend Snippet: Species distribution for homology search of Illumina sequences against the nr database. Species distribution is shown as percentage of the total number of homologous sequences with an E-value cutoff of 1.0E −5 . The most significant hit of each sequence was used for further analysis.

Techniques Used: Sequencing

16) Product Images from "Efficient nonmeiotic allele introgression in livestock using custom endonucleases"

Article Title: Efficient nonmeiotic allele introgression in livestock using custom endonucleases

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1310478110

Sequence analysis of TALEN-stimulated HDR alleles. PCR amplicons flanking the target site (200–250 bp total) derived from TALEN mRNA and oligo-transfected cell populations were sequenced by Illumina sequencing. The total read count ranged from
Figure Legend Snippet: Sequence analysis of TALEN-stimulated HDR alleles. PCR amplicons flanking the target site (200–250 bp total) derived from TALEN mRNA and oligo-transfected cell populations were sequenced by Illumina sequencing. The total read count ranged from

Techniques Used: Sequencing, Polymerase Chain Reaction, Derivative Assay, Transfection

17) Product Images from "High capacity DNA data storage with variable-length Oligonucleotides using repeat accumulate code and hybrid mapping"

Article Title: High capacity DNA data storage with variable-length Oligonucleotides using repeat accumulate code and hybrid mapping

Journal: Journal of Biological Engineering

doi: 10.1186/s13036-019-0211-2

Sequencing result analysis and data recovery. ( A ) The quality value of each base position along the reads. The first half part of the x axis is for reads 1 and the latter half part is for reads 2. ( B ) The error rate of each base position along the reads. The first half part of the distribution is for reads 1 and the latter half part is for reads 2. ( C ) The base content of each base position along the reads. A/T/G/C denote the type of nucleotides and N denotes a lost nucleotide which can be any one of A/T/G/C. The distribution is separated by two reads, note that for (a), (b) and (c), read 1 and read 2 are obtained from randomly sequencing from either the end of each sequence. ( D ) The experimental procedure for data recovery. The amplified and prepared synthetic oligo samples are sequenced using Illumina HiSeq sequencing technology. With five sets of down-sampling trials, different sizes of randomly chosen portions of raw sequence reads are sent to the decoder where the stored files are recovered. ( E ) The number of correctly recovered sequences against the coverage. The black circle markers represent recovered sequences before RA decoding and diamond markers represent recovered sequences after RA decoding. Among the diamond markers, red ones represent partial recovery, while green ones represent full recovery
Figure Legend Snippet: Sequencing result analysis and data recovery. ( A ) The quality value of each base position along the reads. The first half part of the x axis is for reads 1 and the latter half part is for reads 2. ( B ) The error rate of each base position along the reads. The first half part of the distribution is for reads 1 and the latter half part is for reads 2. ( C ) The base content of each base position along the reads. A/T/G/C denote the type of nucleotides and N denotes a lost nucleotide which can be any one of A/T/G/C. The distribution is separated by two reads, note that for (a), (b) and (c), read 1 and read 2 are obtained from randomly sequencing from either the end of each sequence. ( D ) The experimental procedure for data recovery. The amplified and prepared synthetic oligo samples are sequenced using Illumina HiSeq sequencing technology. With five sets of down-sampling trials, different sizes of randomly chosen portions of raw sequence reads are sent to the decoder where the stored files are recovered. ( E ) The number of correctly recovered sequences against the coverage. The black circle markers represent recovered sequences before RA decoding and diamond markers represent recovered sequences after RA decoding. Among the diamond markers, red ones represent partial recovery, while green ones represent full recovery

Techniques Used: Sequencing, Amplification, Sampling

Related Articles

Diagnostic Assay:

Article Title: Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer
Article Snippet: MiRNAs seem to be promising diagnostic biomarkers for colorectal cancer; however, there were multiple studies with conflicting findings. .. In particular we compared the expression of more than 300 miRNAs using Illumina next-generation sequencing.

Clone Assay:

Article Title: Methanogenic Degradation of Long n-Alkanes Requires Fumarate-Dependent Activation
Article Snippet: The positive clones were sent for Sanger sequencing ( ). .. After PCR amplification, the final PCR products were sequenced using Illumina next-generation sequencing in accordance with previously described methods ( ).

Amplification:

Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
Article Snippet: Based on the approach, it is essential for the two termini of target DNA fragment to ligate to two different adaptors, so to permit the binding of two different PCR primers, each complementary to a specific site in the adaptor, for full-length amplification of the target DNA. .. This issue was addressed by elegant designs using forked/Y adaptor (Illumina, US 20100273662 A1) and loop adaptor (NEB, US 20120244525 A1, WO 2012012037 A1) for Illumina next-generation sequencing.

Article Title: Methanogenic Degradation of Long n-Alkanes Requires Fumarate-Dependent Activation
Article Snippet: .. After PCR amplification, the final PCR products were sequenced using Illumina next-generation sequencing in accordance with previously described methods ( ). ..

Article Title: Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus
Article Snippet: Illumina next-generation sequencing Two rounds of PCR were performed on gDNA. .. Amplification was performed in a 50 μl reaction volume, consisting of 10 μl of 5 × HercII buffer, 0.5 μM forward primer, 0.5 μM reverse primer, 50 μM dNTP, 50 mM MgCl2 , 0.5 μl of HercII polymerase and 250 ng of gDNA template.

Article Title: YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs
Article Snippet: .. Illumina next-generation sequencing of the amplified cDNA bands yielded ∼15–16 million raw reads, high percentage ( > 96%) of which were actually mapped to tRNA space (Figure and ). .. This is a significant improvement compared to the conventional RNA-seq method in which the Illumina sequencing reads of BT-474 cDNAs, derived from tRNA region (Figure ), showed only 25% mapping rate to tRNA space (data not shown).

High Throughput Screening Assay:

Article Title: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis
Article Snippet: Expressed sequence tags (ESTs) from cDNA libraries were the first generation of high-throughput transcriptome acquisition using Sanger sequencing technique. .. Short read length from Illumina next-generation sequencing, makes it very difficult, if not impossible to associate a particular splicing event with another, particularly for transcripts containing repetitive elements.

Transduction:

Article Title: RNA-Seq Analysis of Cocos nucifera: Transcriptome Sequencing and De Novo Assembly for Subsequent Functional Genomics Approaches
Article Snippet: Conclusions We applied Illumina next-generation sequencing and de novo assembly to elucidate the coconut transcriptome for the first time. .. Of the 57,304 unigenes, 23,168 were mapped into 215 KEGG pathways, including galactose metabolism, plant-pathogen interaction and plant hormone signal transduction pathways.

Quantitative RT-PCR:

Article Title: Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer
Article Snippet: In particular we compared the expression of more than 300 miRNAs using Illumina next-generation sequencing. .. We identified miRNA expression profiles characteristic for colon and rectal cancers and confirmed miRNA expression differences obtained with this technique with a second unrelated technique, real-time quantitative polymerase chain reaction (RT-qPCR) for relevant miRNAs, thereby, revealing the potential of several miRNAs as new diagnostic biomarkers.

Real-time Polymerase Chain Reaction:

Article Title: Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer
Article Snippet: In particular we compared the expression of more than 300 miRNAs using Illumina next-generation sequencing. .. We identified miRNA expression profiles characteristic for colon and rectal cancers and confirmed miRNA expression differences obtained with this technique with a second unrelated technique, real-time quantitative polymerase chain reaction (RT-qPCR) for relevant miRNAs, thereby, revealing the potential of several miRNAs as new diagnostic biomarkers.

Expressing:

Article Title: Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer
Article Snippet: .. In particular we compared the expression of more than 300 miRNAs using Illumina next-generation sequencing. .. We identified miRNA expression profiles characteristic for colon and rectal cancers and confirmed miRNA expression differences obtained with this technique with a second unrelated technique, real-time quantitative polymerase chain reaction (RT-qPCR) for relevant miRNAs, thereby, revealing the potential of several miRNAs as new diagnostic biomarkers.

Article Title: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis
Article Snippet: RNA sequencing (RNA-seq) arose based on the development of next-generation sequencing techniques and has made transcriptome analysis useful for differential gene expression analysis, alternative gene splicing characterisation, gene mutation and single-nucleotide polymorphism identification and detection of gene fusions [ ]. .. Short read length from Illumina next-generation sequencing, makes it very difficult, if not impossible to associate a particular splicing event with another, particularly for transcripts containing repetitive elements.

Touchdown PCR:

Article Title: Methanogenic Degradation of Long n-Alkanes Requires Fumarate-Dependent Activation
Article Snippet: The extracted DNA was amplified using touchdown PCR by primer sets 515F (5′-GTGCCAGCMGCCGCGG-3′)/907R (5′-CCGTCAATTCMTTTRAGTTT-3′) ( ) for bacteria and 344F (5′-ACGGGGYGCAGCAGGCGCGA-3′)/915R (5′-GTGCTCCCCCGCCAATTCCT-3′) ( ) for archaea. .. After PCR amplification, the final PCR products were sequenced using Illumina next-generation sequencing in accordance with previously described methods ( ).

Modification:

Article Title: SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing
Article Snippet: To speed this revolution in evolutionary genetics, we have developed R estriction site A ssociated D NA (RAD) genotyping, a method that uses Illumina next-generation sequencing to simultaneously discover and score tens to hundreds of thousands of single-nucleotide polymorphism (SNP) markers in hundreds of individuals for minimal investment of resources. .. In this chapter, we describe the core RAD-seq protocol, which can be modified to suit a diversity of evolutionary genetic questions.

Derivative Assay:

Article Title: Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer
Article Snippet: The authors state that further studies identifying the source of tumor derived miRNAs in circulation, including identification of exosomal miRNA content, were required [ ]. .. In particular we compared the expression of more than 300 miRNAs using Illumina next-generation sequencing.

Article Title: YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs
Article Snippet: Illumina next-generation sequencing of the amplified cDNA bands yielded ∼15–16 million raw reads, high percentage ( > 96%) of which were actually mapped to tRNA space (Figure and ). .. This is a significant improvement compared to the conventional RNA-seq method in which the Illumina sequencing reads of BT-474 cDNAs, derived from tRNA region (Figure ), showed only 25% mapping rate to tRNA space (data not shown).

Ligation:

Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
Article Snippet: This issue was addressed by elegant designs using forked/Y adaptor (Illumina, US 20100273662 A1) and loop adaptor (NEB, US 20120244525 A1, WO 2012012037 A1) for Illumina next-generation sequencing. .. Furthermore, the ligation and amplification efficiencies also need to be further improved for the amplification of low abundance or low copy number DNA molecules.

Low Copy Number:

Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
Article Snippet: This issue was addressed by elegant designs using forked/Y adaptor (Illumina, US 20100273662 A1) and loop adaptor (NEB, US 20120244525 A1, WO 2012012037 A1) for Illumina next-generation sequencing. .. Furthermore, the ligation and amplification efficiencies also need to be further improved for the amplification of low abundance or low copy number DNA molecules.

Generated:

Article Title: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis
Article Snippet: For root tissue, we generated 102 334 full-length consensus transcripts, including 73 755 polished HQ and 28 579 LQ transcripts ( ). .. The N50 (N50 length similar to the median contig or assembled sequence length and is defined as the length N for which 50% of all bases in the assembled sequences are in an assembled sequence of length L Illumina next-generation sequencing ( , N50=1 205 nt for 454 assembled transcripts according to reference [ ].).

Article Title: Optimized Illumina PCR-free library preparation for bacterial whole genome sequencing and analysis of factors influencing de novo assembly
Article Snippet: Based on the optimized protocol developed, sequencing libraries with flexible insert sizes and lower reagent costs can be generated. .. Furthermore, increased knowledge about the interplay of sequencing quality, insert size, genomic GC content, read length, sequencing depth and the assembler used will help molecular biologists to set up an optimal experimental and analytical framework with respect to Illumina next-generation sequencing of bacterial genomes.

Sequencing:

Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
Article Snippet: The outcome was not satisfactory due to significant loss of sequence information and inevitable requirement of large amount of DNA input. .. This issue was addressed by elegant designs using forked/Y adaptor (Illumina, US 20100273662 A1) and loop adaptor (NEB, US 20120244525 A1, WO 2012012037 A1) for Illumina next-generation sequencing.

Article Title: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis
Article Snippet: .. The N50 (N50 length similar to the median contig or assembled sequence length and is defined as the length N for which 50% of all bases in the assembled sequences are in an assembled sequence of length L Illumina next-generation sequencing ( , N50=1 205 nt for 454 assembled transcripts according to reference [ ].). .. To compare the transcripts produced by different sequencing platforms, we looked at complete open reading frames (ORFs) from Iso-Seq consensus transcripts and Illumina de novo assembled transcripts aligned against a well-curated full-length protein database, UniProt Knowledgebase (UniProtKB).

Article Title: Methanogenic Degradation of Long n-Alkanes Requires Fumarate-Dependent Activation
Article Snippet: Paragraph title: 16S rRNA gene sequencing. ... After PCR amplification, the final PCR products were sequenced using Illumina next-generation sequencing in accordance with previously described methods ( ).

Article Title: Optimized Illumina PCR-free library preparation for bacterial whole genome sequencing and analysis of factors influencing de novo assembly
Article Snippet: .. Furthermore, increased knowledge about the interplay of sequencing quality, insert size, genomic GC content, read length, sequencing depth and the assembler used will help molecular biologists to set up an optimal experimental and analytical framework with respect to Illumina next-generation sequencing of bacterial genomes. ..

Article Title: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis
Article Snippet: However, the fundamental limitation for both ESTs and RNA-seq based transcriptome characterisation is the incomplete nature of their sequencing products, which require assembly. .. Short read length from Illumina next-generation sequencing, makes it very difficult, if not impossible to associate a particular splicing event with another, particularly for transcripts containing repetitive elements.

Article Title: YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs
Article Snippet: Illumina next-generation sequencing of the amplified cDNA bands yielded ∼15–16 million raw reads, high percentage ( > 96%) of which were actually mapped to tRNA space (Figure and ). .. This is a significant improvement compared to the conventional RNA-seq method in which the Illumina sequencing reads of BT-474 cDNAs, derived from tRNA region (Figure ), showed only 25% mapping rate to tRNA space (data not shown).

Binding Assay:

Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
Article Snippet: Based on the approach, it is essential for the two termini of target DNA fragment to ligate to two different adaptors, so to permit the binding of two different PCR primers, each complementary to a specific site in the adaptor, for full-length amplification of the target DNA. .. This issue was addressed by elegant designs using forked/Y adaptor (Illumina, US 20100273662 A1) and loop adaptor (NEB, US 20120244525 A1, WO 2012012037 A1) for Illumina next-generation sequencing.

RNA Sequencing Assay:

Article Title: YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs
Article Snippet: Illumina next-generation sequencing of the amplified cDNA bands yielded ∼15–16 million raw reads, high percentage ( > 96%) of which were actually mapped to tRNA space (Figure and ). .. This is a significant improvement compared to the conventional RNA-seq method in which the Illumina sequencing reads of BT-474 cDNAs, derived from tRNA region (Figure ), showed only 25% mapping rate to tRNA space (data not shown).

Magnetic Beads:

Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
Article Snippet: This strategy was carried over to SOLiDTM NGS protocol which amplifies DNA fragments on magnetic beads for next-generation sequencing. .. This issue was addressed by elegant designs using forked/Y adaptor (Illumina, US 20100273662 A1) and loop adaptor (NEB, US 20120244525 A1, WO 2012012037 A1) for Illumina next-generation sequencing.

Mutagenesis:

Article Title: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis
Article Snippet: RNA sequencing (RNA-seq) arose based on the development of next-generation sequencing techniques and has made transcriptome analysis useful for differential gene expression analysis, alternative gene splicing characterisation, gene mutation and single-nucleotide polymorphism identification and detection of gene fusions [ ]. .. Short read length from Illumina next-generation sequencing, makes it very difficult, if not impossible to associate a particular splicing event with another, particularly for transcripts containing repetitive elements.

Isolation:

Article Title: YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs
Article Snippet: High specificity, sensitivity and reproducibility of YAMAT-seq Given the success of mature tRNA amplification from BT-474 cells, we further applied the YAMAT-seq method to total RNAs isolated from three additional breast cancer cell lines, MCF-7, SK-BR-3 and BT-20, and each performed in triplicate. .. Illumina next-generation sequencing of the amplified cDNA bands yielded ∼15–16 million raw reads, high percentage ( > 96%) of which were actually mapped to tRNA space (Figure and ).

Purification:

Article Title: Methanogenic Degradation of Long n-Alkanes Requires Fumarate-Dependent Activation
Article Snippet: 16S rRNA genes of the inoculum were amplified using primers 8F/805R ( ) for bacteria and primers 340F/1000R ( ) for archaea, and PCR analyses were performed according to a previous study , which was followed by purification and cloning. .. After PCR amplification, the final PCR products were sequenced using Illumina next-generation sequencing in accordance with previously described methods ( ).

Polymerase Chain Reaction:

Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
Article Snippet: Based on the approach, it is essential for the two termini of target DNA fragment to ligate to two different adaptors, so to permit the binding of two different PCR primers, each complementary to a specific site in the adaptor, for full-length amplification of the target DNA. .. This issue was addressed by elegant designs using forked/Y adaptor (Illumina, US 20100273662 A1) and loop adaptor (NEB, US 20120244525 A1, WO 2012012037 A1) for Illumina next-generation sequencing.

Article Title: Methanogenic Degradation of Long n-Alkanes Requires Fumarate-Dependent Activation
Article Snippet: .. After PCR amplification, the final PCR products were sequenced using Illumina next-generation sequencing in accordance with previously described methods ( ). ..

Article Title: Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus
Article Snippet: .. Illumina next-generation sequencing Two rounds of PCR were performed on gDNA. ..

Next-Generation Sequencing:

Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
Article Snippet: .. This issue was addressed by elegant designs using forked/Y adaptor (Illumina, US 20100273662 A1) and loop adaptor (NEB, US 20120244525 A1, WO 2012012037 A1) for Illumina next-generation sequencing. .. However, the procedures remain tedious and NGS-oriented commercialization has made these strategies cost-ineffective.

Article Title: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis
Article Snippet: .. The N50 (N50 length similar to the median contig or assembled sequence length and is defined as the length N for which 50% of all bases in the assembled sequences are in an assembled sequence of length L Illumina next-generation sequencing ( , N50=1 205 nt for 454 assembled transcripts according to reference [ ].). .. To compare the transcripts produced by different sequencing platforms, we looked at complete open reading frames (ORFs) from Iso-Seq consensus transcripts and Illumina de novo assembled transcripts aligned against a well-curated full-length protein database, UniProt Knowledgebase (UniProtKB).

Article Title: SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing
Article Snippet: .. We presented one application of NGS, RAD genotyping, a focused reduced-representation methodology that uses Illumina next-generation sequencing to simultaneously discover and score tens to hundreds of thousands markers in a very cost-efficient manner. ..

Article Title: Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer
Article Snippet: .. In particular we compared the expression of more than 300 miRNAs using Illumina next-generation sequencing. .. We identified miRNA expression profiles characteristic for colon and rectal cancers and confirmed miRNA expression differences obtained with this technique with a second unrelated technique, real-time quantitative polymerase chain reaction (RT-qPCR) for relevant miRNAs, thereby, revealing the potential of several miRNAs as new diagnostic biomarkers.

Article Title: Methanogenic Degradation of Long n-Alkanes Requires Fumarate-Dependent Activation
Article Snippet: .. After PCR amplification, the final PCR products were sequenced using Illumina next-generation sequencing in accordance with previously described methods ( ). ..

Article Title: Optimized Illumina PCR-free library preparation for bacterial whole genome sequencing and analysis of factors influencing de novo assembly
Article Snippet: .. Furthermore, increased knowledge about the interplay of sequencing quality, insert size, genomic GC content, read length, sequencing depth and the assembler used will help molecular biologists to set up an optimal experimental and analytical framework with respect to Illumina next-generation sequencing of bacterial genomes. ..

Article Title: Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus
Article Snippet: .. Illumina next-generation sequencing Two rounds of PCR were performed on gDNA. ..

Article Title: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis
Article Snippet: .. Short read length from Illumina next-generation sequencing, makes it very difficult, if not impossible to associate a particular splicing event with another, particularly for transcripts containing repetitive elements. ..

Article Title: SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing
Article Snippet: .. To speed this revolution in evolutionary genetics, we have developed R estriction site A ssociated D NA (RAD) genotyping, a method that uses Illumina next-generation sequencing to simultaneously discover and score tens to hundreds of thousands of single-nucleotide polymorphism (SNP) markers in hundreds of individuals for minimal investment of resources. .. In this chapter, we describe the core RAD-seq protocol, which can be modified to suit a diversity of evolutionary genetic questions.

Article Title: RNA-Seq Analysis of Cocos nucifera: Transcriptome Sequencing and De Novo Assembly for Subsequent Functional Genomics Approaches
Article Snippet: .. Conclusions We applied Illumina next-generation sequencing and de novo assembly to elucidate the coconut transcriptome for the first time. .. In total, 57,304 unigenes were obtained, with an average unigene length of 752 bp.

Article Title: YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs
Article Snippet: .. Illumina next-generation sequencing of the amplified cDNA bands yielded ∼15–16 million raw reads, high percentage ( > 96%) of which were actually mapped to tRNA space (Figure and ). .. This is a significant improvement compared to the conventional RNA-seq method in which the Illumina sequencing reads of BT-474 cDNAs, derived from tRNA region (Figure ), showed only 25% mapping rate to tRNA space (data not shown).

Sampling:

Article Title: SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing
Article Snippet: To speed this revolution in evolutionary genetics, we have developed R estriction site A ssociated D NA (RAD) genotyping, a method that uses Illumina next-generation sequencing to simultaneously discover and score tens to hundreds of thousands of single-nucleotide polymorphism (SNP) markers in hundreds of individuals for minimal investment of resources. .. Despite considerable progress, the development of analytical tools remains in its infancy, and further work is needed to fully quantify sampling variance and biases in these data types.

Produced:

Article Title: Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis
Article Snippet: By applying the standard Iso-Seq classification and clustering protocol (see Materials and Methods) on the above ROIs, we produced 115 725 full-length consensus transcripts, including 75 816 polished high-quality (HQ) and 39 909 low-quality (LQ) transcripts for leaf tissue. .. The N50 (N50 length similar to the median contig or assembled sequence length and is defined as the length N for which 50% of all bases in the assembled sequences are in an assembled sequence of length L Illumina next-generation sequencing ( , N50=1 205 nt for 454 assembled transcripts according to reference [ ].).

Concentration Assay:

Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids
Article Snippet: This issue was addressed by elegant designs using forked/Y adaptor (Illumina, US 20100273662 A1) and loop adaptor (NEB, US 20120244525 A1, WO 2012012037 A1) for Illumina next-generation sequencing. .. This is particularly important because large amounts of clinical samples are examined on daily basis and significant portion of those are either of low quantity and/or low quality due to various reasons, including paraffin-embedding, long or improper storage, or at small volume or low concentration.

Marker:

Article Title: SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing
Article Snippet: To speed this revolution in evolutionary genetics, we have developed R estriction site A ssociated D NA (RAD) genotyping, a method that uses Illumina next-generation sequencing to simultaneously discover and score tens to hundreds of thousands of single-nucleotide polymorphism (SNP) markers in hundreds of individuals for minimal investment of resources. .. In addition, we discuss bioinformatic considerations that arise from unique aspects of next-generation sequencing data as compared to traditional marker-based approaches, and we outline some general analytical approaches for RAD-seq and similar data.

Clear Native PAGE:

Article Title: YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs
Article Snippet: As shown in Figure , amplified cDNAs from the total RNAs of different cell lines exhibited similar band patterns on native PAGE, indicating the feasibility and general versatility of YAMAT-seq for the amplification of mature tRNAs among total RNAs from various cells. .. Illumina next-generation sequencing of the amplified cDNA bands yielded ∼15–16 million raw reads, high percentage ( > 96%) of which were actually mapped to tRNA space (Figure and ).

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    Illumina Inc illumina next generation sequencing
    Statistical analysis of a de novo assembly of Cocos nucifera short reads generated by <t>Illumina</t> Hiseq™ 2000 sequencing. The distribution of assembled contigs and unigene lengths is shown (127,952 contigs and 57,304 unigenes were generated in the study).
    Illumina Next Generation Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/illumina next generation sequencing/product/Illumina Inc
    Average 99 stars, based on 44 article reviews
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    Illumina Inc miseq next generation sequencing
    Phylum‐level distributions of bacterial community, determined by <t>Illumina</t> <t>MiSeq</t> sequencing of bee bread from 20 hives, organized by location (on an east–west axis left to right)
    Miseq Next Generation Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Statistical analysis of a de novo assembly of Cocos nucifera short reads generated by Illumina Hiseq™ 2000 sequencing. The distribution of assembled contigs and unigene lengths is shown (127,952 contigs and 57,304 unigenes were generated in the study).

    Journal: PLoS ONE

    Article Title: RNA-Seq Analysis of Cocos nucifera: Transcriptome Sequencing and De Novo Assembly for Subsequent Functional Genomics Approaches

    doi: 10.1371/journal.pone.0059997

    Figure Lengend Snippet: Statistical analysis of a de novo assembly of Cocos nucifera short reads generated by Illumina Hiseq™ 2000 sequencing. The distribution of assembled contigs and unigene lengths is shown (127,952 contigs and 57,304 unigenes were generated in the study).

    Article Snippet: Conclusions We applied Illumina next-generation sequencing and de novo assembly to elucidate the coconut transcriptome for the first time.

    Techniques: Generated, Sequencing

    Phylum‐level distributions of bacterial community, determined by Illumina MiSeq sequencing of bee bread from 20 hives, organized by location (on an east–west axis left to right)

    Journal: Ecology and Evolution

    Article Title: Bacterial communities associated with honeybee food stores are correlated with land use. Bacterial communities associated with honeybee food stores are correlated with land use

    doi: 10.1002/ece3.3999

    Figure Lengend Snippet: Phylum‐level distributions of bacterial community, determined by Illumina MiSeq sequencing of bee bread from 20 hives, organized by location (on an east–west axis left to right)

    Article Snippet: Here, we explore this hypothesis with a 16S rRNA gene amplicon fingerprinting survey using both denaturing gradient gel electrophoresis (DGGE) and Illumina MiSeq next‐generation sequencing (NGS).

    Techniques: Sequencing