sequencing platform Illumina Inc Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Illumina Inc illumina miseq platform
    Schematic representation of the sample preparation (A) and the bioinformatics data handling (B) of the metabarcoded sequencing library. (A) In the first-round PCR amplification, overhanging forward and reverse primers were used to amplify the rDNA 18S. The adapter base pairs provide the target sites for the primers used for sequencing and the random nucleotides (0-3Ns) were inserted between the primers and the adapter to offset the reading frame, therefore amplicons prevent the oversaturation of the <t>MiSeq</t> sequencing channels. The second-round PCR amplification was then performed using overhanging barcoded primers bound to the adapter tags to add indices, as well as the P7 and P5 regions required to bind to the MiSeq flow cell. (B) Text files containing rDNA 18S sequence data (FASTQ files) were generated from the <t>Illumina</t> MiSeq binary raw data outputs, and data analyses were performed using a bespoke modified pipeline in Mothur v1.39.5 software ( Schloss et al., 2009 ) and Illumina MiSeq standard procedures ( Kozich et al., 2013 ) as described in materials and methods section 2.4 .
    Illumina Miseq Platform, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 72122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/illumina miseq platform/product/Illumina Inc
    Average 94 stars, based on 72122 article reviews
    Price from $9.99 to $1999.99
    illumina miseq platform - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    94
    Illumina Inc hiseq 2000 platform
    Validation of RNA-Seq data by quantitative real-time PCR (qRT-PCR).  (A)  Comparison of the expressions profile of eight DEGs determined by Illumina HiSeq™ 2000 sequencing platform and qRT-PCR at 48 h (pi) using ribosomal protein subunit S4 ( rps4 ) as housekeeping gene. Data shown are the mean of triplicates ± SD.  (B)  Correlation of data between RNA-Seq and qRT-PCR techniques.
    Hiseq 2000 Platform, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 24696 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hiseq 2000 platform/product/Illumina Inc
    Average 94 stars, based on 24696 article reviews
    Price from $9.99 to $1999.99
    hiseq 2000 platform - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    95
    Illumina Inc cdna libraries
    Sequence features and validation of circRNAs in M. oryzae . ( a ) Venn diagram showing the number of tissue-preferentially expressed circRNAs in mycelium and conidium of M.oryzae . ( b ) Distribution of circRNAs in genome region of M. oryzae . ( c ) Length distribution of circRNAs. ( d ) An example of M. oryzae circRNAs (mor_circ_04492) shows the validation strategy. Divergent and convergent primers were designed to detect circular <t>RNAs.</t> Sanger sequencing was performed to confirm head-to-tail backsplicing. ( e ) Experimental validation of M. oryzae circRNAs. Divergent primers successfully amplified circRNAs in <t>cDNA</t> but failed in genomic DNA. Amplification for sequence of actin gene was used as a control. The gels were cropped from the same gel, and the full-length gel was supported in Fig. S2 .
    Cdna Libraries, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 95/100, based on 30682 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdna libraries/product/Illumina Inc
    Average 95 stars, based on 30682 article reviews
    Price from $9.99 to $1999.99
    cdna libraries - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    99
    Illumina Inc hiseq 4000 platform
    Evaluation of inter-platform consistency. For 19 cross-platform replicates at 99% CI, 91.89% genes in the BGISEQ-500 datasets showed the expected mapped read count fluctuations using HiSeq 2000 (A). The Spearman correlation analyses revealed high agreement within 19 pair of platform replicates between BGISEQ-500 and HiSeq 2000 (B) (an average Spearman's rho of 0.724 at gene level [top] and 0.948 at species level [bottom]) and between BGISEQ-500 and <t>HiSeq</t> 4000 (C) (an average Spearman's rho of 0.859 at gene level [top] and 0.965 at species level [bottom]).
    Hiseq 4000 Platform, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 5002 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hiseq 4000 platform/product/Illumina Inc
    Average 99 stars, based on 5002 article reviews
    Price from $9.99 to $1999.99
    hiseq 4000 platform - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Illumina Inc nextseq 500 platform
    Gene expression analysis in PBMCs from MOG-AAD patients: Single cell RNA sequencing (inDrop) was performed on an untreated MOG-AAD patient#1 with 2 longitudinal samples, (1) Remission (MOG-AAD#1.2) and (2) Pre-relapse (MOG-AAD#1.3) as described in Materials and Methods. cDNA libraries were sequenced using the Illumina <t>NextSeq</t> 500 platform and analyzed following V3 Indrop criteria. After sequencing the raw BCL files were demultiplexed using bcl2fastq software by illumina ( https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html ). Reads obtained from bcl2fastq were further processed using the single-cell RNA-seq pipeline of the bcbio-nextgen ( https://bcbio-nextgen.readthedocs.io/en/latest/contents/pipelines.html#single-cell-rna-seq ) software suite. The scaled data was further clustered using Seurat and visualized using TSNE ( https://www.biorxiv.org/content/early/2018/11/02/460147 ). A Cluster analysis of a relapse and remission sample. B Differential expression of TNFAIP3 in the relapse and remission sample by single cell sequencing. Digital Gene Expression (DGE) sequencing was performed on an untreated MOG-AAD patient#1 with 3 longitudinal samples, (1) Rm (remission, MOG-AA#1.2), (2) PR (pre-relapse, MOG-AAD#1.3), and (3) R (relapse, MOG-AAD#1.4) as described in Materials and Methods. Raw BCL files generated through sequencing were further de-multiplexed using Picard ( https://github.com/broadinstitute/picard ) and the resulting FASTQ files where aligned to the human reference genome (GRCh38) using the STAR v2.4.2a 52 aligner. Further QC was done using the RNA-seQC 53 and transcript counts were produced using feature Counts function of the Subread package 54 . Data was normalized using the DESeq2 package 55 and the graphs were made using GraphPadPrism version 8.4.2 (464). C TNFAIP3 and NFκβ1 expression by DGE sequencing. NanoString Gene Expression Assay was performed on a MOG-AAD patient#2 with 3 longitudinal samples, (1) untreated R (relapse, MOG-AAD#2.1), (2) 8 months Rm/Ct (mycophenolate mofetil treated at remission, MOG-AAD#2.2), and (3) 11 months Rm/Ct (mycophenolate mofetil treated at remission, MOG-AAD#2.3) as described in Materials and Methods. Data were normalized and analyzed using nSolver software via the geometric mean of included housekeeping genes. The graphs were made using GraphPadPrism version 8.4.2 (464). D TNFAIP3 and TNF-α expression by NanoString Gene Expression Assay. qPCR was performed on CD4+ T cells from 7 MOG-AAD patients with longitudinal samples as described in Materials and Methods. It also included MOG-AAD patient#2 with 3 longitudinal samples as previously used for NanoString gene expression assay. The graphs were made using GraphPadPrism version 8.4.2 (464). E TNFAIP3 expression in MOG-AAD patient#2 by qPCR. F Grouped analysis of TNFAIP3 expression in relapse samples, remission samples and samples treated with corticosteroids by qPCR. Relapse n = 5, Remission n = 5, Steroid n = 4, Ordinary 1-way ANOVA; P = 0.0137.
    Nextseq 500 Platform, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 3734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nextseq 500 platform/product/Illumina Inc
    Average 93 stars, based on 3734 article reviews
    Price from $9.99 to $1999.99
    nextseq 500 platform - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    94
    Illumina Inc 16s rrna gene
    Relative abundance of OTUs from the Sponge Microbiome Project with ≥98% identity to <t>16S</t> <t>rRNA</t> sequences from strains isolated in this study. (A) Information relative to OTUs closely affiliated to isolate Cc27, (B) Information relative to OTUs closely affiliated with isolate Pv91. (C) Information relative to OTUs closely affiliated with isolates Pv86. Vertical bar represents the mean, the hinge represents SEM (Standard Error of Mean), and dots represent outlier values beyond mean.
    16s Rrna Gene, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 7249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/16s rrna gene/product/Illumina Inc
    Average 94 stars, based on 7249 article reviews
    Price from $9.99 to $1999.99
    16s rrna gene - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    94
    Illumina Inc illumina gaiix platform
    DG template preparation workflow. Genomic DNA is digested with Fse I. Index adapters are ligated to the Fse I ends. The DNA fragments are randomly sheared, followed by size selection on agarose gels. DNA fragments of a selected size are end-repaired. A T-tailed adapter is ligated to the repaired ends of the DNA. PCR is carried out with a biotinylated oligonucleotide primer complementary to the Index adapter. DNA fragments labelled with biotin are captured via magnetic beads. The purified DNA fragments are amplified by PCR with <t>Illumina</t> Primers. Amplification products are sequenced on the Illumina <t>GAIIx</t> sequencer. The colored regions in the DNA fragments correspond to the colored regions in the detailed views of the Index and T-tailed adapters in Figure 6 .
    Illumina Gaiix Platform, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 3229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/illumina gaiix platform/product/Illumina Inc
    Average 94 stars, based on 3229 article reviews
    Price from $9.99 to $1999.99
    illumina gaiix platform - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    93
    Illumina Inc genomic dna
    Cellular <t>DNA</t> at the integration region is co-amplified with HPV. A, The location of Brd4, HPV16 DNA, and cellular DNA flanking the chromosome 2 integration site were studied in W12 <t>20861</t> and 20863 (extrachromosomal HPV16) cells, and in HPV-negative NIKS human keratinocyte cells by combined immunofluorescence /fluorescent in situ hybridization. HPV16 DNA is shown in red, and the flanking cellular sequence is shown in green. Brd4 signal is in cyan and nuclei are counterstained with DAPI (blue). In comparable FISH experiments, about 70% 20861 cells contained large focus of signal from the cellular flanking sequence that colocalized with the HPV16 signal. B, Alignment of 20861 WGS data to the human reference genome (hg19) showed focal amplification of cellular sequence at the HPV16 integration site. Histograms represent depth of coverage of aligned reads (quality score threshold, 30). Amplified region is marked by a black horizontal bar; HPV16 genome and cellular genes are represented by green and blue horizontal arrows, respectively. C, WGS reads were aligned to the HPV type 16 isolate 16W12E (AF125673.1) reference genome. Histograms represent copy numbers of viral sequences (blue) and counts of discordant paired-end reads supporting insertional breakpoints (red). The scale (y-axis) of each plot was normalized to maximum read counts. HPV breakpoints are defined further in Table 1 and S1 Table .
    Genomic Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 12613 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genomic dna/product/Illumina Inc
    Average 93 stars, based on 12613 article reviews
    Price from $9.99 to $1999.99
    genomic dna - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    97
    Illumina Inc preparation kit
    Cellular <t>DNA</t> at the integration region is co-amplified with HPV. A, The location of Brd4, HPV16 DNA, and cellular DNA flanking the chromosome 2 integration site were studied in W12 <t>20861</t> and 20863 (extrachromosomal HPV16) cells, and in HPV-negative NIKS human keratinocyte cells by combined immunofluorescence /fluorescent in situ hybridization. HPV16 DNA is shown in red, and the flanking cellular sequence is shown in green. Brd4 signal is in cyan and nuclei are counterstained with DAPI (blue). In comparable FISH experiments, about 70% 20861 cells contained large focus of signal from the cellular flanking sequence that colocalized with the HPV16 signal. B, Alignment of 20861 WGS data to the human reference genome (hg19) showed focal amplification of cellular sequence at the HPV16 integration site. Histograms represent depth of coverage of aligned reads (quality score threshold, 30). Amplified region is marked by a black horizontal bar; HPV16 genome and cellular genes are represented by green and blue horizontal arrows, respectively. C, WGS reads were aligned to the HPV type 16 isolate 16W12E (AF125673.1) reference genome. Histograms represent copy numbers of viral sequences (blue) and counts of discordant paired-end reads supporting insertional breakpoints (red). The scale (y-axis) of each plot was normalized to maximum read counts. HPV breakpoints are defined further in Table 1 and S1 Table .
    Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 97/100, based on 18159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/preparation kit/product/Illumina Inc
    Average 97 stars, based on 18159 article reviews
    Price from $9.99 to $1999.99
    preparation kit - by Bioz Stars, 2020-09
    97/100 stars
      Buy from Supplier

    94
    Illumina Inc rna sequencing
    Cellular <t>DNA</t> at the integration region is co-amplified with HPV. A, The location of Brd4, HPV16 DNA, and cellular DNA flanking the chromosome 2 integration site were studied in W12 <t>20861</t> and 20863 (extrachromosomal HPV16) cells, and in HPV-negative NIKS human keratinocyte cells by combined immunofluorescence /fluorescent in situ hybridization. HPV16 DNA is shown in red, and the flanking cellular sequence is shown in green. Brd4 signal is in cyan and nuclei are counterstained with DAPI (blue). In comparable FISH experiments, about 70% 20861 cells contained large focus of signal from the cellular flanking sequence that colocalized with the HPV16 signal. B, Alignment of 20861 WGS data to the human reference genome (hg19) showed focal amplification of cellular sequence at the HPV16 integration site. Histograms represent depth of coverage of aligned reads (quality score threshold, 30). Amplified region is marked by a black horizontal bar; HPV16 genome and cellular genes are represented by green and blue horizontal arrows, respectively. C, WGS reads were aligned to the HPV type 16 isolate 16W12E (AF125673.1) reference genome. Histograms represent copy numbers of viral sequences (blue) and counts of discordant paired-end reads supporting insertional breakpoints (red). The scale (y-axis) of each plot was normalized to maximum read counts. HPV breakpoints are defined further in Table 1 and S1 Table .
    Rna Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 3143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna sequencing/product/Illumina Inc
    Average 94 stars, based on 3143 article reviews
    Price from $9.99 to $1999.99
    rna sequencing - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    95
    Illumina Inc rna seq libraries
    Graphical representation of C. maritima growth under two salinity conditions (bars represent SD). The growth in freshwater is represented by filled squares, whereas the marine growth is represented as the solid circles. Statistical significance between the curves was assessed using a permutation test to compare growth curves. The test was applied to the colony diameter until effects on the growth were most apparent, that is, 16–19 d after inoculation. The pair-wise comparisons between the fungi samples were statistically nonsignificant ( P -value 0.0999). The arrows indicate the time of growth from where the <t>RNA</t> isolation was performed for either the <t>transcriptome</t> or the RT-qPCR analysis.
    Rna Seq Libraries, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 95/100, based on 14521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna seq libraries/product/Illumina Inc
    Average 95 stars, based on 14521 article reviews
    Price from $9.99 to $1999.99
    rna seq libraries - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    94
    Illumina Inc cbot cluster generation system
    Graphical representation of C. maritima growth under two salinity conditions (bars represent SD). The growth in freshwater is represented by filled squares, whereas the marine growth is represented as the solid circles. Statistical significance between the curves was assessed using a permutation test to compare growth curves. The test was applied to the colony diameter until effects on the growth were most apparent, that is, 16–19 d after inoculation. The pair-wise comparisons between the fungi samples were statistically nonsignificant ( P -value 0.0999). The arrows indicate the time of growth from where the <t>RNA</t> isolation was performed for either the <t>transcriptome</t> or the RT-qPCR analysis.
    Cbot Cluster Generation System, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 3721 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cbot cluster generation system/product/Illumina Inc
    Average 94 stars, based on 3721 article reviews
    Price from $9.99 to $1999.99
    cbot cluster generation system - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    Image Search Results


    Schematic representation of the sample preparation (A) and the bioinformatics data handling (B) of the metabarcoded sequencing library. (A) In the first-round PCR amplification, overhanging forward and reverse primers were used to amplify the rDNA 18S. The adapter base pairs provide the target sites for the primers used for sequencing and the random nucleotides (0-3Ns) were inserted between the primers and the adapter to offset the reading frame, therefore amplicons prevent the oversaturation of the MiSeq sequencing channels. The second-round PCR amplification was then performed using overhanging barcoded primers bound to the adapter tags to add indices, as well as the P7 and P5 regions required to bind to the MiSeq flow cell. (B) Text files containing rDNA 18S sequence data (FASTQ files) were generated from the Illumina MiSeq binary raw data outputs, and data analyses were performed using a bespoke modified pipeline in Mothur v1.39.5 software ( Schloss et al., 2009 ) and Illumina MiSeq standard procedures ( Kozich et al., 2013 ) as described in materials and methods section 2.4 .

    Journal: bioRxiv

    Article Title: A novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

    doi: 10.1101/801175

    Figure Lengend Snippet: Schematic representation of the sample preparation (A) and the bioinformatics data handling (B) of the metabarcoded sequencing library. (A) In the first-round PCR amplification, overhanging forward and reverse primers were used to amplify the rDNA 18S. The adapter base pairs provide the target sites for the primers used for sequencing and the random nucleotides (0-3Ns) were inserted between the primers and the adapter to offset the reading frame, therefore amplicons prevent the oversaturation of the MiSeq sequencing channels. The second-round PCR amplification was then performed using overhanging barcoded primers bound to the adapter tags to add indices, as well as the P7 and P5 regions required to bind to the MiSeq flow cell. (B) Text files containing rDNA 18S sequence data (FASTQ files) were generated from the Illumina MiSeq binary raw data outputs, and data analyses were performed using a bespoke modified pipeline in Mothur v1.39.5 software ( Schloss et al., 2009 ) and Illumina MiSeq standard procedures ( Kozich et al., 2013 ) as described in materials and methods section 2.4 .

    Article Snippet: In conclusion, we describe for the first time the use of metabarcoded DNA sequencing using an Illumina MiSeq platform to quantify P. falciparum and P. vivax, and demonstrate its accuracy on malaria-positive samples.

    Techniques: Sample Prep, Sequencing, Polymerase Chain Reaction, Amplification, Generated, Modification, Software

    Schematic work-flow of C. retropictus transcriptome using Illumina sequencing, de novo analysis, and annotation. The visceral mass transcriptome of C. retropictus was obtained using an Illumina HiSeq 2500 platform. The raw reads were pre-processed using the Sickle software tool (quality: 20, length: 40) and Fastq_filter software to obtain clean reads. Using Trinity (K-mer: 25; minimum contig length: 200) de novo assembler and TGICL clustering, the clean reads were processed to unigene sequences. Subsequently, the unigene sequences were blasted against public databases including PANM, Unigene, COG, GO, and KEGG for functional annotation. The SSRs were detected within the unigene sequences using MISA software.

    Journal: Genes

    Article Title: Transcriptomic Analysis of the Endangered Neritid Species Clithon retropictus: De Novo Assembly, Functional Annotation, and Marker Discovery

    doi: 10.3390/genes7070035

    Figure Lengend Snippet: Schematic work-flow of C. retropictus transcriptome using Illumina sequencing, de novo analysis, and annotation. The visceral mass transcriptome of C. retropictus was obtained using an Illumina HiSeq 2500 platform. The raw reads were pre-processed using the Sickle software tool (quality: 20, length: 40) and Fastq_filter software to obtain clean reads. Using Trinity (K-mer: 25; minimum contig length: 200) de novo assembler and TGICL clustering, the clean reads were processed to unigene sequences. Subsequently, the unigene sequences were blasted against public databases including PANM, Unigene, COG, GO, and KEGG for functional annotation. The SSRs were detected within the unigene sequences using MISA software.

    Article Snippet: Finally, the library preparations were sequenced on an Illumina HiSeq 2500 platform at the sequencing facility of GnC Company (Daejeon, South Korea) with the generation of 100-base pair (bp) PE-reads.

    Techniques: Flow Cytometry, Sequencing, Software, Functional Assay

    Correlation, dissimilarity, and change in α-diversity between stored or transported and fresh fecal samples. Sequencing data (dataset A) used to plot this figure was generated by using the Illumina HiSeq 4000 platform. “/T” represents transportation groups. a The α-diversity of the freshly extracted samples and corresponding stored samples. b – d The Euclidean distance, Bray-Curtis dissimilarity, and Spearman correlation coefficient between the stored samples and corresponding freshly extracted ones. One asterisk indicates significant difference ( p

    Journal: Microbiome

    Article Title: A novel affordable reagent for room temperature storage and transport of fecal samples for metagenomic analyses

    doi: 10.1186/s40168-018-0429-0

    Figure Lengend Snippet: Correlation, dissimilarity, and change in α-diversity between stored or transported and fresh fecal samples. Sequencing data (dataset A) used to plot this figure was generated by using the Illumina HiSeq 4000 platform. “/T” represents transportation groups. a The α-diversity of the freshly extracted samples and corresponding stored samples. b – d The Euclidean distance, Bray-Curtis dissimilarity, and Spearman correlation coefficient between the stored samples and corresponding freshly extracted ones. One asterisk indicates significant difference ( p

    Article Snippet: Library construction and sequencing Sequencing was performed using either the Illumina HiSeq platform or the BGISEQ-500 platform, which was recently validated and benchmarked against the Illumina HiSeq platform [ ].

    Techniques: Sequencing, Generated

    High-throughput workflow for the preparation of plasmid DNA libraries for NGS. Plasmid DNA samples are isolated from bacteria cells using a high-throughput plasmid isolation method on the CyBio ® FeliX robot. All steps performed using the FeliX platform are highlighted with a grey outline. The isolated plasmid samples are tested for the presence of genomic DNA (gDNA), prior to library preparation, using the Labcyte Echo ® . All steps performed using the Labcyte Echo ® are highlighted with a shaded grey box. If samples are free from gDNA, they are diluted to 0.4 ng/μl in H 2 O. If gDNA is detected, the samples are sonicated prior to re-testing in the gDNA QC assay. Using reagents from the Nextera XT kit, a tagmentation reaction is performed on all samples under the optimised conditions, followed by neutralization of the reaction. Unique combinations of index primers are added to all samples via 12 PCR cycles, followed by magnetic bead purification of the PCR products. The concentration of the purified dsDNA is then determined using the PicoGreen ® reagent assay and the libraries are pooled to give a final concentration of 4–10 nM, in a minimum volume of 15 μl. The average fragment size of the pooled libraries is measured using the Fragment Analyzer before being sequenced on the Illumina ® MiSeq system.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: High-throughput workflow for the preparation of plasmid DNA libraries for NGS. Plasmid DNA samples are isolated from bacteria cells using a high-throughput plasmid isolation method on the CyBio ® FeliX robot. All steps performed using the FeliX platform are highlighted with a grey outline. The isolated plasmid samples are tested for the presence of genomic DNA (gDNA), prior to library preparation, using the Labcyte Echo ® . All steps performed using the Labcyte Echo ® are highlighted with a shaded grey box. If samples are free from gDNA, they are diluted to 0.4 ng/μl in H 2 O. If gDNA is detected, the samples are sonicated prior to re-testing in the gDNA QC assay. Using reagents from the Nextera XT kit, a tagmentation reaction is performed on all samples under the optimised conditions, followed by neutralization of the reaction. Unique combinations of index primers are added to all samples via 12 PCR cycles, followed by magnetic bead purification of the PCR products. The concentration of the purified dsDNA is then determined using the PicoGreen ® reagent assay and the libraries are pooled to give a final concentration of 4–10 nM, in a minimum volume of 15 μl. The average fragment size of the pooled libraries is measured using the Fragment Analyzer before being sequenced on the Illumina ® MiSeq system.

    Article Snippet: 3.3 Optimisation of plasmid DNA library preparation for NGS using Design of Experiments For sequencing of plasmid DNA using the Illumina ® MiSeq platform, a library of fragments with a mean size of between 200 and 400 bp, each with a concentration of 0.5–5 ng/μl is required.

    Techniques: High Throughput Screening Assay, Plasmid Preparation, Next-Generation Sequencing, Isolation, Sonication, Neutralization, Polymerase Chain Reaction, Purification, Concentration Assay

    Next generation sequencing of plasmid DNA libraries, prepared using a miniaturised method with the Nextera XT library preparation kit. 96 plasmid DNA libraries were prepared for NGS with the Nextera XT library preparation kit, using optimised conditions (12.5 min incubation, 50 nl sample, 1.8x magnetic bead solution). ( A ) After purification, the samples were quantified in the PicoGreen ® dsDNA quantification assay. The data show that 75/92 samples have a concentration within the desired range (0.5–5 ng/μl), with an average concentration of 1.1 ng/μl. These samples were pooled, with a final concentration of 6.64 nM and run on the Fragment Analyzer ( B ) and ( C ). All fragments in the pooled libraries are of the desired size (200–400 bp). ( D ) The pooled library was sequenced on the Illumina ® MiSeq system (2 × 150 method). The mean sequence quality (Phred) scores are plotted for each sample. For all samples, the sequence quality (Phred) score was > 30 for more than 85% of the base pairs, indicating that all samples passed the QC criteria.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Next generation sequencing of plasmid DNA libraries, prepared using a miniaturised method with the Nextera XT library preparation kit. 96 plasmid DNA libraries were prepared for NGS with the Nextera XT library preparation kit, using optimised conditions (12.5 min incubation, 50 nl sample, 1.8x magnetic bead solution). ( A ) After purification, the samples were quantified in the PicoGreen ® dsDNA quantification assay. The data show that 75/92 samples have a concentration within the desired range (0.5–5 ng/μl), with an average concentration of 1.1 ng/μl. These samples were pooled, with a final concentration of 6.64 nM and run on the Fragment Analyzer ( B ) and ( C ). All fragments in the pooled libraries are of the desired size (200–400 bp). ( D ) The pooled library was sequenced on the Illumina ® MiSeq system (2 × 150 method). The mean sequence quality (Phred) scores are plotted for each sample. For all samples, the sequence quality (Phred) score was > 30 for more than 85% of the base pairs, indicating that all samples passed the QC criteria.

    Article Snippet: 3.3 Optimisation of plasmid DNA library preparation for NGS using Design of Experiments For sequencing of plasmid DNA using the Illumina ® MiSeq platform, a library of fragments with a mean size of between 200 and 400 bp, each with a concentration of 0.5–5 ng/μl is required.

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Incubation, Purification, Concentration Assay, Sequencing

    Illumina HiSeq2000 QC of one representative sample. A) Raw quality scores (FASTQ) of reads from one bisulfite treated library (G12; lane 6). B) Graph showing the representation of each base at each position in a 50 base read. Notice the very low level of Cytosine residues compared to the high content of Adenosine residues; non-bisulifte converted DNA shows approximately equal base composition at each site. One metric of C-T conversion rate is the ratio of C residues at bases 1–10 and 40–50. In the graph above, this ratio is about 1, as expected. In RRBS libraries, a large increase in Guanidine at positions 2 and 3 (~95% of reads have GG at these positions) should also be observed.

    Journal: BMC Genomics

    Article Title: BisQC: an operational pipeline for multiplexed bisulfite sequencing

    doi: 10.1186/1471-2164-15-290

    Figure Lengend Snippet: Illumina HiSeq2000 QC of one representative sample. A) Raw quality scores (FASTQ) of reads from one bisulfite treated library (G12; lane 6). B) Graph showing the representation of each base at each position in a 50 base read. Notice the very low level of Cytosine residues compared to the high content of Adenosine residues; non-bisulifte converted DNA shows approximately equal base composition at each site. One metric of C-T conversion rate is the ratio of C residues at bases 1–10 and 40–50. In the graph above, this ratio is about 1, as expected. In RRBS libraries, a large increase in Guanidine at positions 2 and 3 (~95% of reads have GG at these positions) should also be observed.

    Article Snippet: Massively parallel (next generation) sequencing We used the Illumina HiSeq2000 platform, with single-end 50 cycle sequencing.

    Techniques:

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

    Journal: mSystems

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

    doi: 10.1128/mSystems.00039-15

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

    Article Snippet: Final products of the SOP were shipped to JCVI, pooled, and sequenced on the Illumina MiSeq (two 300-bp paired end [PE] sequencing reads), Illumina HiSeq (two 100-bp PE), Illumina NextSeq (two 150-bp PE), or Ion Torrent (318 Chip 400-bp kit) platforms.

    Techniques: Sequencing, Western Blot

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

    Journal: mSystems

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

    doi: 10.1128/mSystems.00039-15

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

    Article Snippet: Final products of the SOP were shipped to JCVI, pooled, and sequenced on the Illumina MiSeq (two 300-bp paired end [PE] sequencing reads), Illumina HiSeq (two 100-bp PE), Illumina NextSeq (two 150-bp PE), or Ion Torrent (318 Chip 400-bp kit) platforms.

    Techniques: Sequencing, Generated

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

    Journal: mSystems

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

    doi: 10.1128/mSystems.00039-15

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

    Article Snippet: Final products of the SOP were shipped to JCVI, pooled, and sequenced on the Illumina MiSeq (two 300-bp paired end [PE] sequencing reads), Illumina HiSeq (two 100-bp PE), Illumina NextSeq (two 150-bp PE), or Ion Torrent (318 Chip 400-bp kit) platforms.

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

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

    Journal: mSystems

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

    doi: 10.1128/mSystems.00039-15

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

    Article Snippet: Final products of the SOP were shipped to JCVI, pooled, and sequenced on the Illumina MiSeq (two 300-bp paired end [PE] sequencing reads), Illumina HiSeq (two 100-bp PE), Illumina NextSeq (two 150-bp PE), or Ion Torrent (318 Chip 400-bp kit) platforms.

    Techniques: Sequencing

    —Data analysis overview. DNA and RNA were isolated from A. viridis adult polyps sampled from a natural seawater pH gradient (at normal seawater pH 8.2, and at low seawater pH 7.9 and 7.6) off Vulcano Island, Sicily—Italy. Only one polyp (pH 8.2) was used for DNA extraction and was subjected to paired-end sequencing on the Illumina HiSeq2500 platform. Sequencing reads were assembled into a draft genome reference. Subsequently, repeat-enriched regions, including transposable elements were identified and annotated in this assembly. Nine polyps were used for small RNA library preparation and sequencing on the SOLiD 5500xl platform. Sequencing reads were further used for novel miRNA discovery and description of putative piRNA reads.

    Journal: Genome Biology and Evolution

    Article Title: Elucidating the Small Regulatory RNA Repertoire of the Sea Anemone Anemonia viridis Based on Whole Genome and Small RNA Sequencing

    doi: 10.1093/gbe/evy003

    Figure Lengend Snippet: —Data analysis overview. DNA and RNA were isolated from A. viridis adult polyps sampled from a natural seawater pH gradient (at normal seawater pH 8.2, and at low seawater pH 7.9 and 7.6) off Vulcano Island, Sicily—Italy. Only one polyp (pH 8.2) was used for DNA extraction and was subjected to paired-end sequencing on the Illumina HiSeq2500 platform. Sequencing reads were assembled into a draft genome reference. Subsequently, repeat-enriched regions, including transposable elements were identified and annotated in this assembly. Nine polyps were used for small RNA library preparation and sequencing on the SOLiD 5500xl platform. Sequencing reads were further used for novel miRNA discovery and description of putative piRNA reads.

    Article Snippet: Genome Reference Assembly and Search for Repeat-Enriched Regions Total DNA from a single A. viridis polyp (normal seawater conditions, pH 8.2) was extracted and subjected to whole genome sequencing on the Illumina HiSeq2500 platform ( ).

    Techniques: Isolation, DNA Extraction, Sequencing

    Frequency distribution of the contig sizes from two Gynostemma species. The frequency distribution of contig sizes resulting from Illumina HiSeq™ 2000 sequencing, as assembled using Trinity.

    Journal: Molecules

    Article Title: Characterization of Global Transcriptome Using Illumina Paired-End Sequencing and Development of EST-SSR Markers in Two Species of Gynostemma (Cucurbitaceae)

    doi: 10.3390/molecules201219758

    Figure Lengend Snippet: Frequency distribution of the contig sizes from two Gynostemma species. The frequency distribution of contig sizes resulting from Illumina HiSeq™ 2000 sequencing, as assembled using Trinity.

    Article Snippet: With the development of next-generation sequencing (NGS), it has become possible to generate large numbers of transcriptomic datasets for nonmodel organisms [ ] using various platforms such as Roche 454, Illumina HiSeq, and Applied Biosystems SOLiD.

    Techniques: Sequencing

    Organization of fungal rDNA locus and regions targeted by oligonucleotide probe and primers. Primers UNI1, UNI2, and Cspecies, and probe are used in the qPCR strategy. Primers Fseq and Rseq are used to generate amplicons for sequencing. Cgla, C . glabrata ; Ctro, C . tropicalis ; Cpar, C . parapsilosis ; Ckru, C . krusei ; Calb, C . albicans . Locus depiction is not to scale. Primer sequences are given in Table 1 .

    Journal: PLoS ONE

    Article Title: Complementary Amplicon-Based Genomic Approaches for the Study of Fungal Communities in Humans

    doi: 10.1371/journal.pone.0116705

    Figure Lengend Snippet: Organization of fungal rDNA locus and regions targeted by oligonucleotide probe and primers. Primers UNI1, UNI2, and Cspecies, and probe are used in the qPCR strategy. Primers Fseq and Rseq are used to generate amplicons for sequencing. Cgla, C . glabrata ; Ctro, C . tropicalis ; Cpar, C . parapsilosis ; Ckru, C . krusei ; Calb, C . albicans . Locus depiction is not to scale. Primer sequences are given in Table 1 .

    Article Snippet: Equal concentrations of amplicons from each individual fecal sample were pooled into a final solution and submitted for Illumina library construction using the TruSeq Nano kit (Illumina, San Diego, CA) and sequencing on an Illumina MiSeq high-throughput sequencing platform using the 2 × 150 bp paired-end version 2 MiSeq Reagent Kit (Illumina) by the University of Minnesota Genomics Center.

    Techniques: Real-time Polymerase Chain Reaction, Sequencing

    —Examples of sequence alignments between Bemisia tabaci “Peru” MEAM1 mtDNA COI haplotype gene region, published “MEAM2” mtDNA COI gene region, and NGS candidate NUMT sequences identified from KX234913, KY951449, and the KY951452 individuals. ( A ) C-terminal region of a Peruvian MEAM1 B. tabaci (KY951450) mtDNA COI gene region showing putative stop codon (black shaded “*” symbol), as well as the B. tabaci “MEAM2” haplotype from Japan (AJ550177), and examples NGS candidate NUMT sequences from the Peruvian B. tabaci MEAM1 (KX234914) with matching SNPs (indicated by red boxes) that matched the Japan MEAM2 haplotype (AJ550177). Deletion of a “T” base (indicated by red triangle) resulted in a frameshift mutation and the loss of the putative mtDNA COI gene stop codon. ( B ) Internal stop codons (at positions 904S906) detected in candidate NUMT sequences (KY951452_NUMT-01, KY951452_NUMT-02) from the Peruvian MEAM1 individual (KY951452) MiSeq generated DNA fragments when compared with the Peruvian B. tabaci MEAM1 (KX234914) mtDNA COI gene. Stop codons detected in NUMT sequences were the result of a single nucleotide base change at position 906 from a “T” to an “A.” Candidate NUMT sequences were also compared with the Peruvian MEAM2 haplotype (KY951454) obtained via PCR and Sanger sequencing of the same MEAM1 individual (KY951452). Nucleotide positions based on the mtDNA COI gene are provided. Amino acid translation based on the invertebrate mitochondrial genetic codes (Translational Table_5). Significant changes between amino acids are highlighted.

    Journal: Genome Biology and Evolution

    Article Title: The Trouble with MEAM2: Implications of Pseudogenes on Species Delimitation in the Globally Invasive Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species Complex

    doi: 10.1093/gbe/evx173

    Figure Lengend Snippet: —Examples of sequence alignments between Bemisia tabaci “Peru” MEAM1 mtDNA COI haplotype gene region, published “MEAM2” mtDNA COI gene region, and NGS candidate NUMT sequences identified from KX234913, KY951449, and the KY951452 individuals. ( A ) C-terminal region of a Peruvian MEAM1 B. tabaci (KY951450) mtDNA COI gene region showing putative stop codon (black shaded “*” symbol), as well as the B. tabaci “MEAM2” haplotype from Japan (AJ550177), and examples NGS candidate NUMT sequences from the Peruvian B. tabaci MEAM1 (KX234914) with matching SNPs (indicated by red boxes) that matched the Japan MEAM2 haplotype (AJ550177). Deletion of a “T” base (indicated by red triangle) resulted in a frameshift mutation and the loss of the putative mtDNA COI gene stop codon. ( B ) Internal stop codons (at positions 904S906) detected in candidate NUMT sequences (KY951452_NUMT-01, KY951452_NUMT-02) from the Peruvian MEAM1 individual (KY951452) MiSeq generated DNA fragments when compared with the Peruvian B. tabaci MEAM1 (KX234914) mtDNA COI gene. Stop codons detected in NUMT sequences were the result of a single nucleotide base change at position 906 from a “T” to an “A.” Candidate NUMT sequences were also compared with the Peruvian MEAM2 haplotype (KY951454) obtained via PCR and Sanger sequencing of the same MEAM1 individual (KY951452). Nucleotide positions based on the mtDNA COI gene are provided. Amino acid translation based on the invertebrate mitochondrial genetic codes (Translational Table_5). Significant changes between amino acids are highlighted.

    Article Snippet: We identified low copy genome fragments through the Illumina MiSeq sequencing platform in MEAM1 individuals that matched unique MEAM2 SNPs ( ).

    Techniques: Sequencing, Next-Generation Sequencing, Mutagenesis, Generated, Polymerase Chain Reaction

    The sequence coverage of germline variants and the length of the indels which were identified in one sequence platform. Read depth in Illumina for variants unique to BGISEQ-500 ( a ) read depth in BGI for variants unique to Illumina ( b ). The distribution of the length (number of bases) of the indels that were identified in both sequencing platforms or unique to the HiSeq X Ten or BGISEQ-500 data is plotted ( c ).

    Journal: PLoS ONE

    Article Title: Germline and somatic variant identification using BGISEQ-500 and HiSeq X Ten whole genome sequencing

    doi: 10.1371/journal.pone.0190264

    Figure Lengend Snippet: The sequence coverage of germline variants and the length of the indels which were identified in one sequence platform. Read depth in Illumina for variants unique to BGISEQ-500 ( a ) read depth in BGI for variants unique to Illumina ( b ). The distribution of the length (number of bases) of the indels that were identified in both sequencing platforms or unique to the HiSeq X Ten or BGISEQ-500 data is plotted ( c ).

    Article Snippet: In this study we performed whole genome sequencing of three malignant pleural mesothelioma and matched normal samples using a new platform, the BGISEQ-500, and compared the results obtained with Illumina HiSeq X Ten.

    Techniques: Sequencing

    The growth of SRA data categorized by project types, and sequencing platforms. (A) The growth of the number of SRA studies categorized by project types. The number of studies are double that of the previous year. (B) The growth of the number of SRA experiments categorized by sequencing platforms. Over 200,000 experiments are submitted under approximately 14,000 studies. The experiments using Illumina HiSeq 2000 are dramatically increasing.

    Journal: PLoS ONE

    Article Title: Experimental Design-Based Functional Mining and Characterization of High-Throughput Sequencing Data in the Sequence Read Archive

    doi: 10.1371/journal.pone.0077910

    Figure Lengend Snippet: The growth of SRA data categorized by project types, and sequencing platforms. (A) The growth of the number of SRA studies categorized by project types. The number of studies are double that of the previous year. (B) The growth of the number of SRA experiments categorized by sequencing platforms. Over 200,000 experiments are submitted under approximately 14,000 studies. The experiments using Illumina HiSeq 2000 are dramatically increasing.

    Article Snippet: The top 3 of most used sequencer are Illumina HiSeq 2000, Illumina Genome Analyzer II, and 454 GS FLX Titanium (92,888, 42,274, and 19,463 experiments, respectively), and experiments with new sequencing platforms such as Complete Genomics, Helicos HeliScope, PacBio RS, and Ion Torrent PGM are also archived (1133, 437, 431, and 304 experiments, respectively).

    Techniques: Sequencing

    Combined 16S sequencing results for the gut microbiomes of sympatric colonies of A. echinatior , A. octospinosus , and A. volcanus using both Roche 454 and Illumina Miseq 16S sequencing. OTU heat maps show the relative abundances (rarefied number of reads)

    Journal: Applied and Environmental Microbiology

    Article Title: Acromyrmex Leaf-Cutting Ants Have Simple Gut Microbiota with Nitrogen-Fixing Potential

    doi: 10.1128/AEM.00961-15

    Figure Lengend Snippet: Combined 16S sequencing results for the gut microbiomes of sympatric colonies of A. echinatior , A. octospinosus , and A. volcanus using both Roche 454 and Illumina Miseq 16S sequencing. OTU heat maps show the relative abundances (rarefied number of reads)

    Article Snippet: Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform .

    Techniques: Sequencing

    Schematic overview of microbiome bioinformatic analysis workflow. The hypervariable V4 region of 16S rDNA from tick samples was sequenced and split by barcode with Illumina MiSeq. Resulting paired-end reads were joined and the primer region was removed. Reads were filtered by amplicon length and aligned to SILVA as the reference database. After removal of chimeras, reads were clustered into operational taxonomic units (OTU) and taxonomically classified. Finally, an OTU-table was created and results were visualized

    Journal: Parasites & Vectors

    Article Title: Combination of microbiome analysis and serodiagnostics to assess the risk of pathogen transmission by ticks to humans and animals in central Germany

    doi: 10.1186/s13071-018-3240-7

    Figure Lengend Snippet: Schematic overview of microbiome bioinformatic analysis workflow. The hypervariable V4 region of 16S rDNA from tick samples was sequenced and split by barcode with Illumina MiSeq. Resulting paired-end reads were joined and the primer region was removed. Reads were filtered by amplicon length and aligned to SILVA as the reference database. After removal of chimeras, reads were clustered into operational taxonomic units (OTU) and taxonomically classified. Finally, an OTU-table was created and results were visualized

    Article Snippet: All samples were diluted to the same molarity, pooled together, spiked with an internal control (15% PhiX) and paired-end sequenced on the MiSeq Illumina platform using a flow cell with V2 chemistry (500 cycles).

    Techniques: Amplification

    Validation of RNA-Seq data by quantitative real-time PCR (qRT-PCR).  (A)  Comparison of the expressions profile of eight DEGs determined by Illumina HiSeq™ 2000 sequencing platform and qRT-PCR at 48 h (pi) using ribosomal protein subunit S4 ( rps4 ) as housekeeping gene. Data shown are the mean of triplicates ± SD.  (B)  Correlation of data between RNA-Seq and qRT-PCR techniques.

    Journal: Frontiers in Immunology

    Article Title: Transcriptomic Profiles of Senegalese Sole Infected With Nervous Necrosis Virus Reassortants Presenting Different Degree of Virulence

    doi: 10.3389/fimmu.2018.01626

    Figure Lengend Snippet: Validation of RNA-Seq data by quantitative real-time PCR (qRT-PCR). (A) Comparison of the expressions profile of eight DEGs determined by Illumina HiSeq™ 2000 sequencing platform and qRT-PCR at 48 h (pi) using ribosomal protein subunit S4 ( rps4 ) as housekeeping gene. Data shown are the mean of triplicates ± SD. (B) Correlation of data between RNA-Seq and qRT-PCR techniques.

    Article Snippet: Therefore, samples from 48 h p.i. were selected for RNA-Seq analysis (threefold replicated), being sequenced using Illumina HiSeq™ 2000 platform.

    Techniques: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Sequencing

    Sequence features and validation of circRNAs in M. oryzae . ( a ) Venn diagram showing the number of tissue-preferentially expressed circRNAs in mycelium and conidium of M.oryzae . ( b ) Distribution of circRNAs in genome region of M. oryzae . ( c ) Length distribution of circRNAs. ( d ) An example of M. oryzae circRNAs (mor_circ_04492) shows the validation strategy. Divergent and convergent primers were designed to detect circular RNAs. Sanger sequencing was performed to confirm head-to-tail backsplicing. ( e ) Experimental validation of M. oryzae circRNAs. Divergent primers successfully amplified circRNAs in cDNA but failed in genomic DNA. Amplification for sequence of actin gene was used as a control. The gels were cropped from the same gel, and the full-length gel was supported in Fig. S2 .

    Journal: Scientific Reports

    Article Title: Genome-wide Identification and characterization of circular RNAs in the rice blast fungus Magnaporthe oryzae

    doi: 10.1038/s41598-018-25242-w

    Figure Lengend Snippet: Sequence features and validation of circRNAs in M. oryzae . ( a ) Venn diagram showing the number of tissue-preferentially expressed circRNAs in mycelium and conidium of M.oryzae . ( b ) Distribution of circRNAs in genome region of M. oryzae . ( c ) Length distribution of circRNAs. ( d ) An example of M. oryzae circRNAs (mor_circ_04492) shows the validation strategy. Divergent and convergent primers were designed to detect circular RNAs. Sanger sequencing was performed to confirm head-to-tail backsplicing. ( e ) Experimental validation of M. oryzae circRNAs. Divergent primers successfully amplified circRNAs in cDNA but failed in genomic DNA. Amplification for sequence of actin gene was used as a control. The gels were cropped from the same gel, and the full-length gel was supported in Fig. S2 .

    Article Snippet: By using an mRNA-Seq sample preparation kit (Illumina, San Diego, USA), the remaining RNAs were used to construct cDNA libraries, which were subsequently used to perform circRNA sequencing by an Illumina Hiseq2500 platform.

    Techniques: Sequencing, Amplification

    Evaluation of inter-platform consistency. For 19 cross-platform replicates at 99% CI, 91.89% genes in the BGISEQ-500 datasets showed the expected mapped read count fluctuations using HiSeq 2000 (A). The Spearman correlation analyses revealed high agreement within 19 pair of platform replicates between BGISEQ-500 and HiSeq 2000 (B) (an average Spearman's rho of 0.724 at gene level [top] and 0.948 at species level [bottom]) and between BGISEQ-500 and HiSeq 4000 (C) (an average Spearman's rho of 0.859 at gene level [top] and 0.965 at species level [bottom]).

    Journal: GigaScience

    Article Title: Assessment of the cPAS-based BGISEQ-500 platform for metagenomic sequencing

    doi: 10.1093/gigascience/gix133

    Figure Lengend Snippet: Evaluation of inter-platform consistency. For 19 cross-platform replicates at 99% CI, 91.89% genes in the BGISEQ-500 datasets showed the expected mapped read count fluctuations using HiSeq 2000 (A). The Spearman correlation analyses revealed high agreement within 19 pair of platform replicates between BGISEQ-500 and HiSeq 2000 (B) (an average Spearman's rho of 0.724 at gene level [top] and 0.948 at species level [bottom]) and between BGISEQ-500 and HiSeq 4000 (C) (an average Spearman's rho of 0.859 at gene level [top] and 0.965 at species level [bottom]).

    Article Snippet: Cross-platform consistency was evaluated by comparing 20 pairwise replicates on the BGISEQ-500 platform vs the Illumina HiSeq 2000 platform and the Illumina HiSeq 4000 platform.

    Techniques:

    A, GC content distributions of genes that differed significantly in abundance between platforms. Density curves showing a comparison of GC content distributions of the total 9.9 million IGC genes (blue), all 349 479 highly reproducible (HR) genes (green), and all 11 350 genes that differed significantly in abundance between the 2 platforms (red line). B, Two-dimensional plot showing the GC content distribution of genes that differed significantly in abundance between the BGISEQ-500 and HiSeq 2000 platforms. The x-axis indicates the GC content of genes, the y-axis indicates fold-changes of gene relative abundance (RA), which is calculated by log10 transformed mean RA in the HiSeq 2000 datasets/mean RA in the BGISEQ-500 datasets. C, D, Density histograms showing the coefficients of a robust linear model for relative abundance of genes from the top 20 species and their GC content for genes that differed significantly in abundance between the 2 platforms (C) and for all HR genes (D). D, E, Density curves (E) and 2-dimensional plot (F) showing the GC content distributions of HR genes that differed significantly in abundance between the BGISEQ-500 and Hiseq 4000 platforms.

    Journal: GigaScience

    Article Title: Assessment of the cPAS-based BGISEQ-500 platform for metagenomic sequencing

    doi: 10.1093/gigascience/gix133

    Figure Lengend Snippet: A, GC content distributions of genes that differed significantly in abundance between platforms. Density curves showing a comparison of GC content distributions of the total 9.9 million IGC genes (blue), all 349 479 highly reproducible (HR) genes (green), and all 11 350 genes that differed significantly in abundance between the 2 platforms (red line). B, Two-dimensional plot showing the GC content distribution of genes that differed significantly in abundance between the BGISEQ-500 and HiSeq 2000 platforms. The x-axis indicates the GC content of genes, the y-axis indicates fold-changes of gene relative abundance (RA), which is calculated by log10 transformed mean RA in the HiSeq 2000 datasets/mean RA in the BGISEQ-500 datasets. C, D, Density histograms showing the coefficients of a robust linear model for relative abundance of genes from the top 20 species and their GC content for genes that differed significantly in abundance between the 2 platforms (C) and for all HR genes (D). D, E, Density curves (E) and 2-dimensional plot (F) showing the GC content distributions of HR genes that differed significantly in abundance between the BGISEQ-500 and Hiseq 4000 platforms.

    Article Snippet: Cross-platform consistency was evaluated by comparing 20 pairwise replicates on the BGISEQ-500 platform vs the Illumina HiSeq 2000 platform and the Illumina HiSeq 4000 platform.

    Techniques: Transformation Assay

    Gene expression analysis in PBMCs from MOG-AAD patients: Single cell RNA sequencing (inDrop) was performed on an untreated MOG-AAD patient#1 with 2 longitudinal samples, (1) Remission (MOG-AAD#1.2) and (2) Pre-relapse (MOG-AAD#1.3) as described in Materials and Methods. cDNA libraries were sequenced using the Illumina NextSeq 500 platform and analyzed following V3 Indrop criteria. After sequencing the raw BCL files were demultiplexed using bcl2fastq software by illumina ( https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html ). Reads obtained from bcl2fastq were further processed using the single-cell RNA-seq pipeline of the bcbio-nextgen ( https://bcbio-nextgen.readthedocs.io/en/latest/contents/pipelines.html#single-cell-rna-seq ) software suite. The scaled data was further clustered using Seurat and visualized using TSNE ( https://www.biorxiv.org/content/early/2018/11/02/460147 ). A Cluster analysis of a relapse and remission sample. B Differential expression of TNFAIP3 in the relapse and remission sample by single cell sequencing. Digital Gene Expression (DGE) sequencing was performed on an untreated MOG-AAD patient#1 with 3 longitudinal samples, (1) Rm (remission, MOG-AA#1.2), (2) PR (pre-relapse, MOG-AAD#1.3), and (3) R (relapse, MOG-AAD#1.4) as described in Materials and Methods. Raw BCL files generated through sequencing were further de-multiplexed using Picard ( https://github.com/broadinstitute/picard ) and the resulting FASTQ files where aligned to the human reference genome (GRCh38) using the STAR v2.4.2a 52 aligner. Further QC was done using the RNA-seQC 53 and transcript counts were produced using feature Counts function of the Subread package 54 . Data was normalized using the DESeq2 package 55 and the graphs were made using GraphPadPrism version 8.4.2 (464). C TNFAIP3 and NFκβ1 expression by DGE sequencing. NanoString Gene Expression Assay was performed on a MOG-AAD patient#2 with 3 longitudinal samples, (1) untreated R (relapse, MOG-AAD#2.1), (2) 8 months Rm/Ct (mycophenolate mofetil treated at remission, MOG-AAD#2.2), and (3) 11 months Rm/Ct (mycophenolate mofetil treated at remission, MOG-AAD#2.3) as described in Materials and Methods. Data were normalized and analyzed using nSolver software via the geometric mean of included housekeeping genes. The graphs were made using GraphPadPrism version 8.4.2 (464). D TNFAIP3 and TNF-α expression by NanoString Gene Expression Assay. qPCR was performed on CD4+ T cells from 7 MOG-AAD patients with longitudinal samples as described in Materials and Methods. It also included MOG-AAD patient#2 with 3 longitudinal samples as previously used for NanoString gene expression assay. The graphs were made using GraphPadPrism version 8.4.2 (464). E TNFAIP3 expression in MOG-AAD patient#2 by qPCR. F Grouped analysis of TNFAIP3 expression in relapse samples, remission samples and samples treated with corticosteroids by qPCR. Relapse n = 5, Remission n = 5, Steroid n = 4, Ordinary 1-way ANOVA; P = 0.0137.

    Journal: Scientific Reports

    Article Title: Identification of TNFAIP3 as relapse biomarker and potential therapeutic target for MOG antibody associated diseases

    doi: 10.1038/s41598-020-69182-w

    Figure Lengend Snippet: Gene expression analysis in PBMCs from MOG-AAD patients: Single cell RNA sequencing (inDrop) was performed on an untreated MOG-AAD patient#1 with 2 longitudinal samples, (1) Remission (MOG-AAD#1.2) and (2) Pre-relapse (MOG-AAD#1.3) as described in Materials and Methods. cDNA libraries were sequenced using the Illumina NextSeq 500 platform and analyzed following V3 Indrop criteria. After sequencing the raw BCL files were demultiplexed using bcl2fastq software by illumina ( https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html ). Reads obtained from bcl2fastq were further processed using the single-cell RNA-seq pipeline of the bcbio-nextgen ( https://bcbio-nextgen.readthedocs.io/en/latest/contents/pipelines.html#single-cell-rna-seq ) software suite. The scaled data was further clustered using Seurat and visualized using TSNE ( https://www.biorxiv.org/content/early/2018/11/02/460147 ). A Cluster analysis of a relapse and remission sample. B Differential expression of TNFAIP3 in the relapse and remission sample by single cell sequencing. Digital Gene Expression (DGE) sequencing was performed on an untreated MOG-AAD patient#1 with 3 longitudinal samples, (1) Rm (remission, MOG-AA#1.2), (2) PR (pre-relapse, MOG-AAD#1.3), and (3) R (relapse, MOG-AAD#1.4) as described in Materials and Methods. Raw BCL files generated through sequencing were further de-multiplexed using Picard ( https://github.com/broadinstitute/picard ) and the resulting FASTQ files where aligned to the human reference genome (GRCh38) using the STAR v2.4.2a 52 aligner. Further QC was done using the RNA-seQC 53 and transcript counts were produced using feature Counts function of the Subread package 54 . Data was normalized using the DESeq2 package 55 and the graphs were made using GraphPadPrism version 8.4.2 (464). C TNFAIP3 and NFκβ1 expression by DGE sequencing. NanoString Gene Expression Assay was performed on a MOG-AAD patient#2 with 3 longitudinal samples, (1) untreated R (relapse, MOG-AAD#2.1), (2) 8 months Rm/Ct (mycophenolate mofetil treated at remission, MOG-AAD#2.2), and (3) 11 months Rm/Ct (mycophenolate mofetil treated at remission, MOG-AAD#2.3) as described in Materials and Methods. Data were normalized and analyzed using nSolver software via the geometric mean of included housekeeping genes. The graphs were made using GraphPadPrism version 8.4.2 (464). D TNFAIP3 and TNF-α expression by NanoString Gene Expression Assay. qPCR was performed on CD4+ T cells from 7 MOG-AAD patients with longitudinal samples as described in Materials and Methods. It also included MOG-AAD patient#2 with 3 longitudinal samples as previously used for NanoString gene expression assay. The graphs were made using GraphPadPrism version 8.4.2 (464). E TNFAIP3 expression in MOG-AAD patient#2 by qPCR. F Grouped analysis of TNFAIP3 expression in relapse samples, remission samples and samples treated with corticosteroids by qPCR. Relapse n = 5, Remission n = 5, Steroid n = 4, Ordinary 1-way ANOVA; P = 0.0137.

    Article Snippet: 3,000 cells from a cell suspension comprising of CMCs from the 2 samples were isolated into droplets that contained lysis buffer. cDNA libraries were sequenced using the Illumina NextSeq 500 platform and analyzed following V3 Indrop criteria.

    Techniques: Expressing, RNA Sequencing Assay, Sequencing, Software, Generated, Produced, Real-time Polymerase Chain Reaction

    Relative abundance of OTUs from the Sponge Microbiome Project with ≥98% identity to 16S rRNA sequences from strains isolated in this study. (A) Information relative to OTUs closely affiliated to isolate Cc27, (B) Information relative to OTUs closely affiliated with isolate Pv91. (C) Information relative to OTUs closely affiliated with isolates Pv86. Vertical bar represents the mean, the hinge represents SEM (Standard Error of Mean), and dots represent outlier values beyond mean.

    Journal: Frontiers in Microbiology

    Article Title: In Search of Alternative Antibiotic Drugs: Quorum-Quenching Activity in Sponges and their Bacterial Isolates

    doi: 10.3389/fmicb.2016.00416

    Figure Lengend Snippet: Relative abundance of OTUs from the Sponge Microbiome Project with ≥98% identity to 16S rRNA sequences from strains isolated in this study. (A) Information relative to OTUs closely affiliated to isolate Cc27, (B) Information relative to OTUs closely affiliated with isolate Pv91. (C) Information relative to OTUs closely affiliated with isolates Pv86. Vertical bar represents the mean, the hinge represents SEM (Standard Error of Mean), and dots represent outlier values beyond mean.

    Article Snippet: The V4 region of the 16S rRNA gene was amplified using the primers 515F and 806R and sequenced using the HiSeq2500 platform (Illumina) (Caporaso et al., ).

    Techniques: Isolation

    Relative abundance of OTUs from the Sponge Microbiome Project with ≥98% identity to 16S rRNA sequences from strains isolated in this study . (A) Information relative to OTUs closely affiliated to isolate Ss68, (B) Information relative to OTUs closely affiliated with isolate Ac17. Vertical bar represents the mean, the hinge represents SEM (Standard Error of Mean) and dots represent outlier values beyond mean.

    Journal: Frontiers in Microbiology

    Article Title: In Search of Alternative Antibiotic Drugs: Quorum-Quenching Activity in Sponges and their Bacterial Isolates

    doi: 10.3389/fmicb.2016.00416

    Figure Lengend Snippet: Relative abundance of OTUs from the Sponge Microbiome Project with ≥98% identity to 16S rRNA sequences from strains isolated in this study . (A) Information relative to OTUs closely affiliated to isolate Ss68, (B) Information relative to OTUs closely affiliated with isolate Ac17. Vertical bar represents the mean, the hinge represents SEM (Standard Error of Mean) and dots represent outlier values beyond mean.

    Article Snippet: The V4 region of the 16S rRNA gene was amplified using the primers 515F and 806R and sequenced using the HiSeq2500 platform (Illumina) (Caporaso et al., ).

    Techniques: Isolation

    DG template preparation workflow. Genomic DNA is digested with Fse I. Index adapters are ligated to the Fse I ends. The DNA fragments are randomly sheared, followed by size selection on agarose gels. DNA fragments of a selected size are end-repaired. A T-tailed adapter is ligated to the repaired ends of the DNA. PCR is carried out with a biotinylated oligonucleotide primer complementary to the Index adapter. DNA fragments labelled with biotin are captured via magnetic beads. The purified DNA fragments are amplified by PCR with Illumina Primers. Amplification products are sequenced on the Illumina GAIIx sequencer. The colored regions in the DNA fragments correspond to the colored regions in the detailed views of the Index and T-tailed adapters in Figure 6 .

    Journal: BMC Genomics

    Article Title: Digital genotyping of sorghum - a diverse plant species with a large repeat-rich genome

    doi: 10.1186/1471-2164-14-448

    Figure Lengend Snippet: DG template preparation workflow. Genomic DNA is digested with Fse I. Index adapters are ligated to the Fse I ends. The DNA fragments are randomly sheared, followed by size selection on agarose gels. DNA fragments of a selected size are end-repaired. A T-tailed adapter is ligated to the repaired ends of the DNA. PCR is carried out with a biotinylated oligonucleotide primer complementary to the Index adapter. DNA fragments labelled with biotin are captured via magnetic beads. The purified DNA fragments are amplified by PCR with Illumina Primers. Amplification products are sequenced on the Illumina GAIIx sequencer. The colored regions in the DNA fragments correspond to the colored regions in the detailed views of the Index and T-tailed adapters in Figure 6 .

    Article Snippet: After sequencing on the Illumina GAIIx platform, the raw sequencing reads are processed and deconvoluted into individual groups by barcode.

    Techniques: Selection, Polymerase Chain Reaction, Magnetic Beads, Purification, Amplification

    Cellular DNA at the integration region is co-amplified with HPV. A, The location of Brd4, HPV16 DNA, and cellular DNA flanking the chromosome 2 integration site were studied in W12 20861 and 20863 (extrachromosomal HPV16) cells, and in HPV-negative NIKS human keratinocyte cells by combined immunofluorescence /fluorescent in situ hybridization. HPV16 DNA is shown in red, and the flanking cellular sequence is shown in green. Brd4 signal is in cyan and nuclei are counterstained with DAPI (blue). In comparable FISH experiments, about 70% 20861 cells contained large focus of signal from the cellular flanking sequence that colocalized with the HPV16 signal. B, Alignment of 20861 WGS data to the human reference genome (hg19) showed focal amplification of cellular sequence at the HPV16 integration site. Histograms represent depth of coverage of aligned reads (quality score threshold, 30). Amplified region is marked by a black horizontal bar; HPV16 genome and cellular genes are represented by green and blue horizontal arrows, respectively. C, WGS reads were aligned to the HPV type 16 isolate 16W12E (AF125673.1) reference genome. Histograms represent copy numbers of viral sequences (blue) and counts of discordant paired-end reads supporting insertional breakpoints (red). The scale (y-axis) of each plot was normalized to maximum read counts. HPV breakpoints are defined further in Table 1 and S1 Table .

    Journal: PLoS Genetics

    Article Title: HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression

    doi: 10.1371/journal.pgen.1007179

    Figure Lengend Snippet: Cellular DNA at the integration region is co-amplified with HPV. A, The location of Brd4, HPV16 DNA, and cellular DNA flanking the chromosome 2 integration site were studied in W12 20861 and 20863 (extrachromosomal HPV16) cells, and in HPV-negative NIKS human keratinocyte cells by combined immunofluorescence /fluorescent in situ hybridization. HPV16 DNA is shown in red, and the flanking cellular sequence is shown in green. Brd4 signal is in cyan and nuclei are counterstained with DAPI (blue). In comparable FISH experiments, about 70% 20861 cells contained large focus of signal from the cellular flanking sequence that colocalized with the HPV16 signal. B, Alignment of 20861 WGS data to the human reference genome (hg19) showed focal amplification of cellular sequence at the HPV16 integration site. Histograms represent depth of coverage of aligned reads (quality score threshold, 30). Amplified region is marked by a black horizontal bar; HPV16 genome and cellular genes are represented by green and blue horizontal arrows, respectively. C, WGS reads were aligned to the HPV type 16 isolate 16W12E (AF125673.1) reference genome. Histograms represent copy numbers of viral sequences (blue) and counts of discordant paired-end reads supporting insertional breakpoints (red). The scale (y-axis) of each plot was normalized to maximum read counts. HPV breakpoints are defined further in Table 1 and S1 Table .

    Article Snippet: To define the amplification at the HPV16 integration site in 20861 cells, 2 x 150 bp paired-end libraries were generated from total genomic DNA and subjected to WGS using the HiSeq 4000 platform (Illumina Genome Network).

    Techniques: Amplification, Immunofluorescence, In Situ Hybridization, Sequencing, Fluorescence In Situ Hybridization

    20861 cells have multiple, rearranged copies of HPV16 at the integration site. A, Southern blot analysis of 20861 and 20863 W12 cell lines. Genomic DNA was digested with either HindIII (H; does not cut HPV16 DNA) or BamH1 (B; a single cut in HPV16 DNA). DNA fragments were separated by electrophoresis, transferred to membranes and probed with 32P-labeled HPV16 DNA. Arrows indicate relaxed circle (I), unit length linear (II), and supercoiled HPV16 genomes (III). Viral copy number was quantified by comparison to 10 pg linearized HPV16 DNA. The position of BamHI and HindIII cleavage sites spanning the integrated locus (chr2:28,561,317–28,639,445; hg19) and expected band sizes from Southern blot are shown in B . B, Diagram of the integration locus of HPV16 in 20861 cells. APOT analysis showed that HPV16 was integrated into chr2 p23.2 and expresses an E6/E7 fusion transcript that splices to an exon located at 28,595,425–28,595,675 (hg19). The wavy line underneath the expanded viral genome represents the fusion transcript. Primer positions are denoted by black horizontal arrows. Green represents viral sequences, and blue represents host-derived sequences. Splice donor (viral) and acceptor (host) sites are indicated below.

    Journal: PLoS Genetics

    Article Title: HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression

    doi: 10.1371/journal.pgen.1007179

    Figure Lengend Snippet: 20861 cells have multiple, rearranged copies of HPV16 at the integration site. A, Southern blot analysis of 20861 and 20863 W12 cell lines. Genomic DNA was digested with either HindIII (H; does not cut HPV16 DNA) or BamH1 (B; a single cut in HPV16 DNA). DNA fragments were separated by electrophoresis, transferred to membranes and probed with 32P-labeled HPV16 DNA. Arrows indicate relaxed circle (I), unit length linear (II), and supercoiled HPV16 genomes (III). Viral copy number was quantified by comparison to 10 pg linearized HPV16 DNA. The position of BamHI and HindIII cleavage sites spanning the integrated locus (chr2:28,561,317–28,639,445; hg19) and expected band sizes from Southern blot are shown in B . B, Diagram of the integration locus of HPV16 in 20861 cells. APOT analysis showed that HPV16 was integrated into chr2 p23.2 and expresses an E6/E7 fusion transcript that splices to an exon located at 28,595,425–28,595,675 (hg19). The wavy line underneath the expanded viral genome represents the fusion transcript. Primer positions are denoted by black horizontal arrows. Green represents viral sequences, and blue represents host-derived sequences. Splice donor (viral) and acceptor (host) sites are indicated below.

    Article Snippet: To define the amplification at the HPV16 integration site in 20861 cells, 2 x 150 bp paired-end libraries were generated from total genomic DNA and subjected to WGS using the HiSeq 4000 platform (Illumina Genome Network).

    Techniques: Southern Blot, Electrophoresis, Labeling, Derivative Assay

    Analysis of the 20861 integration site by molecular combing. A, Representative image of a DNA fiber containing the Type III HPV16 integration site in 20861 cells. The HPV16 DNA signal is shown in green, and the DNA backbone was visualized with a single-stranded DNA (ssDNA) antibody, shown in red. The map shows the breakpoint of HPV16 in the E2 ORF in the 20861 Type III integration site. B, Scatter plot showing the number of HPV16 genomes per individual DNA fiber. Average signal count per fiber and SD is shown, along with total number of fiber counts. Data are from two replicates. C-D, Scatter plots showing measured HPV16 signal length (C), and measured length of the interspersing space between HPV16 signals from every fiber (D). Measurements were based on the conversion 1 μm = 2 kb. Average measured length in kb and SD is shown, along with total number of fibers counted. Data are from two replicates. E, Two-color fiber-FISH in 20861 cells using DNA probes against HPV16 (shown in red) and the amplified cellular sequence identified from whole genome sequencing (shown in green) confirm the organization of the 20861 Type III integration site as predicted from sequence analysis. Data are from two biological replicates.

    Journal: PLoS Genetics

    Article Title: HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression

    doi: 10.1371/journal.pgen.1007179

    Figure Lengend Snippet: Analysis of the 20861 integration site by molecular combing. A, Representative image of a DNA fiber containing the Type III HPV16 integration site in 20861 cells. The HPV16 DNA signal is shown in green, and the DNA backbone was visualized with a single-stranded DNA (ssDNA) antibody, shown in red. The map shows the breakpoint of HPV16 in the E2 ORF in the 20861 Type III integration site. B, Scatter plot showing the number of HPV16 genomes per individual DNA fiber. Average signal count per fiber and SD is shown, along with total number of fiber counts. Data are from two replicates. C-D, Scatter plots showing measured HPV16 signal length (C), and measured length of the interspersing space between HPV16 signals from every fiber (D). Measurements were based on the conversion 1 μm = 2 kb. Average measured length in kb and SD is shown, along with total number of fibers counted. Data are from two replicates. E, Two-color fiber-FISH in 20861 cells using DNA probes against HPV16 (shown in red) and the amplified cellular sequence identified from whole genome sequencing (shown in green) confirm the organization of the 20861 Type III integration site as predicted from sequence analysis. Data are from two biological replicates.

    Article Snippet: To define the amplification at the HPV16 integration site in 20861 cells, 2 x 150 bp paired-end libraries were generated from total genomic DNA and subjected to WGS using the HiSeq 4000 platform (Illumina Genome Network).

    Techniques: Fluorescence In Situ Hybridization, Amplification, Sequencing

    Super-enhancer markers are enriched over the viral URR in 20861, but not other integrated W12 sub-clones. Chromatin immunoprecipitation (ChIP) was performed in 20861, 20863, 20831 and 20862 cells using antibodies against Brd4 and H3K27ac. A, Map of linearized HPV16 genome showing primer positions (denoted by black horizontal arrows) for the upstream regulatory region (URR), L1 and E2. B, ChIP DNA samples were analyzed by real-time qPCR using primers against target promoters, indicated in panel A. ChIP signals were expressed as the percentage of immunoprecipitated chromatin DNA relative to the total amount of input chromatin (% Input). CCND2 and FOSL1 were included as positive controls for super-enhancer loci; IGLL5 was included as a negative control for Brd4 binding in these cells. C, To account for variations in viral copy number between W12 cells, ChIP signals were expressed as binding per single-copy genome. Background signal at each locus (measured by no-antibody controls) was subtracted from corresponding ChIP signals. Average binding levels were calculated from three independent experiments. Error bars represent SD. Note that similar experiments were previously conducted on 20861 and 20863 cells (using different datasets) [ 22 ].

    Journal: PLoS Genetics

    Article Title: HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression

    doi: 10.1371/journal.pgen.1007179

    Figure Lengend Snippet: Super-enhancer markers are enriched over the viral URR in 20861, but not other integrated W12 sub-clones. Chromatin immunoprecipitation (ChIP) was performed in 20861, 20863, 20831 and 20862 cells using antibodies against Brd4 and H3K27ac. A, Map of linearized HPV16 genome showing primer positions (denoted by black horizontal arrows) for the upstream regulatory region (URR), L1 and E2. B, ChIP DNA samples were analyzed by real-time qPCR using primers against target promoters, indicated in panel A. ChIP signals were expressed as the percentage of immunoprecipitated chromatin DNA relative to the total amount of input chromatin (% Input). CCND2 and FOSL1 were included as positive controls for super-enhancer loci; IGLL5 was included as a negative control for Brd4 binding in these cells. C, To account for variations in viral copy number between W12 cells, ChIP signals were expressed as binding per single-copy genome. Background signal at each locus (measured by no-antibody controls) was subtracted from corresponding ChIP signals. Average binding levels were calculated from three independent experiments. Error bars represent SD. Note that similar experiments were previously conducted on 20861 and 20863 cells (using different datasets) [ 22 ].

    Article Snippet: To define the amplification at the HPV16 integration site in 20861 cells, 2 x 150 bp paired-end libraries were generated from total genomic DNA and subjected to WGS using the HiSeq 4000 platform (Illumina Genome Network).

    Techniques: Clone Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Binding Assay

    Graphical representation of C. maritima growth under two salinity conditions (bars represent SD). The growth in freshwater is represented by filled squares, whereas the marine growth is represented as the solid circles. Statistical significance between the curves was assessed using a permutation test to compare growth curves. The test was applied to the colony diameter until effects on the growth were most apparent, that is, 16–19 d after inoculation. The pair-wise comparisons between the fungi samples were statistically nonsignificant ( P -value 0.0999). The arrows indicate the time of growth from where the RNA isolation was performed for either the transcriptome or the RT-qPCR analysis.

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Comparative Transcriptome Analysis of the Cosmopolitan Marine Fungus Corollospora maritima Under Two Physiological Conditions

    doi: 10.1534/g3.115.019620

    Figure Lengend Snippet: Graphical representation of C. maritima growth under two salinity conditions (bars represent SD). The growth in freshwater is represented by filled squares, whereas the marine growth is represented as the solid circles. Statistical significance between the curves was assessed using a permutation test to compare growth curves. The test was applied to the colony diameter until effects on the growth were most apparent, that is, 16–19 d after inoculation. The pair-wise comparisons between the fungi samples were statistically nonsignificant ( P -value 0.0999). The arrows indicate the time of growth from where the RNA isolation was performed for either the transcriptome or the RT-qPCR analysis.

    Article Snippet: cDNA library construction and whole transcriptome sequencing The RNA-seq libraries were prepared from total RNA using the Illumina TruSeq RNA Sample Preparation Kit following the vendor instructions.

    Techniques: Isolation, Quantitative RT-PCR