pcr primers Illumina Inc Search Results


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  • 99
    Illumina Inc illumina miseq platform
    Phylogeny of 34 most abundant operational taxonomic units (OTUs) of ur e C (corresponding to the species level). ur e C reads obtained by amplicon sequencing on the <t>Illumina</t> <t>MiSeq</t> platform were clustered into species-level OTUs with ≥91% sequence identity, and a phylogenetic tree was constructed by the maximum likelihood method with the Jones-Taylor-Thornton model by means of the ur e C sequence of Canavalia ensiformis (M65260) as an outgroup. Branching points that support probability > 80% in the bootstrap analyses (based on 500 replicates) are shown as filled circles. The heatmap shows the relative abundance of species-level OTUs of ure C in soil samples 1 to 7. The scale bar represents 20% sequence divergence. Nucleotide sequence accession numbers are indicated in parentheses.
    Illumina Miseq Platform, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 74683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc pcr amplification
    Targeted validation of promoter mutations a , Targeted sequencing validation of selected promoter mutations in 47 patients from the ExomePlus cohort with <t>Illumina</t> TruSeq Custom Amplicon panel (TSCA)-targeted sequencing technology. b , Validation rate of promoter mutations calculated as validated mutations over all sequenced and powered mutations. c , Median detection sensitivity at mutated sites for significantly mutated promoters. Each point indicates a single mutated position. d , <t>PCR-MiSeq</t> for the FOXA1 promoter locus for 126 patients with sufficient coverage for mutation calling from the original ExomePlus cohort. Three out of four mutations validated in experiment (green and red bars). PCR-MiSeq for 140 patients included but not covered in original ExomePlus experiment and 64 additional tumours yielded three novel mutations in each set (light and dark blue bars). No germline mutations at this site were detected in normal samples.
    Pcr Amplification, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 12844 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc 16s rrna gene
    Hh infection induces IL-22 dependent dysbiosis (A) Quantification of Hh colonization density in the cecal content. n=9-11. (B) Chao and Shannon diversity indices from <t>16S</t> <t>rRNA</t> sequencing data obtained from fecal pellets of uninfected mice and Hh infected mice treated with control or anti-IL-22 antibody as indicated. (C) Relative abundance of indicated taxa from groups described in B. (D) Relative abundance of indicated taxa as determined by taxa specific 16s rRNA qPCR from groups described in B.
    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
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    94
    Illumina Inc illumina adapters
    Overview of Experimental Methods (A) Schematic of the phenotypes studied. (B) Schematic of the PBAL method. Cells are lysed in 96-well plates to release genomic DNA (gDNA) and then subjected to bisulfite conversion that simultaneously converts and shears the gDNA into fragments. Random hexamers are then used to regenerate double-stranded DNA that is then end-repaired, a-tailed, and ligated with indexed adapters before low-cycle PCR amplification. Libraries are then pooled and sequenced on an <t>Illumina</t> sequencing platform. See also Figures S1 and S2 .
    Illumina Adapters, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 11530 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc eco real time pcr system
    Relative expression of BRCA2 ▼3 and ∆3 transcripts in six c.68‐7T > A carriers and 12 normal controls by quantitative <t>PCR.</t> The boxplots (displaying low, Q1, median, Q3, and high values) show <t>qPCR</t> levels of ▼3 and ∆3 transcripts in carriers and controls. Values are normalized to GUSB mRNA and expressed as fold difference relative to pooled control cDNAs using the ∆ΔCq method (see Materials and Methods). The analysis shows in carriers a statistically significant increase of the relative level of ∆3 transcripts compared to controls (2.98 vs. 0.97; p
    Eco Real Time Pcr System, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 4649 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc nextera xt index kit
    Graphical summary of the SL-seq protocol. (A) cDNA generation with Superscript III and a primer that is partially random (7 nucleotides, grey), and partially fixed (yellow). Consequent degradation of the RNA strand with RNAse H, leaving a single stranded DNA molecule. (B) Second strand synthesis of SL-containing DNA molecules with Klenow fragment and a primer that is complementary with the SL (dark blue). (C + D) PCR for amplification and addition of adapter motives (red and purple) making the library compatible with the <t>Nextera</t> XT index kit (Illumina). (E + F) Final PCR adapter extension, indexing (orange and light blue) and amplification of the library fragments with the primers of the Nextera XT index kit. Dark Blue: SL/ complementary with SL, light grey: RNA, dark grey: DNA, green: poly-A tail. Other colors: primer and adapters sequences.
    Nextera Xt Index Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 4434 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc illumina sequencing adapters
    Experimental design. GFP reporter expression was driven by the EF-1α promoter and potentially modulated by a variable 8mer inserted into the human IQGAP1 3′UTR. The 5′UTR of the GFP reporter contains an intron. Expression of dsRed was used to control for transcriptional noise at the reporter locus, and was driven by the PGK promoter. The flippase recombination target (FRT) site allows this plasmid to undergo site-specific recombination in HEK293-TRex-FLP cells, such that only cells that integrate this construct at the intended locus via the FRT site gain hygromycin resistance. After integration, cells with normal transcriptional activity at the reporter locus (as determined by dsRed levels), and that are potentially undergoing differential post-transcriptional regulation (as determined by GFP levels), were isolated via fluorescence activated cell sorting (FACS). From FACS isolated sub-populations, the portion of the 3′UTR containing the variable 8mer was PCR amplified, thereby adding <t>Illumina</t> adapter sequences, and allowing 8mers in each sorted population to be identified and quantified by Illumina sequencing
    Illumina Sequencing Adapters, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 5299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Illumina Inc dna libraries
    Size selection of the Nextera <t>DNA</t> libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge <t>PCR</t> to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.
    Dna Libraries, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 95/100, based on 11879 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc pcr primers
    Expression levels of 11 PCGs in the nymph (A), pupa (B) and adult (C) of <t>MED</t> whiteflies. Real-Time <t>PCR</t> was used to detect genes expression levels. The x-axis shows the different PCGs, and the y-axis represents the mRNA expression level of different developing stages compared to each control (β-actin).
    Pcr Primers, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 2104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc barcodes
    Vector Contributions and Clonal Diversity of EF1-α, MSCV, and SFFV Barcoded Vectors (A) The percentage contribution of each vector (EF1-α, MSCV, and SFFV) to total transduced hematopoiesis in granulocytes, B cells, and T cells purified from the blood of animals ZJ41 and ZJ48 over time post-transplantation. The read number of each vector’s library ID in a sample over the total read numbers for all library IDs in the sample ×100 is plotted. (B) Number of unique <t>barcodes</t> for each vector (EF1-α, MSCV, and SFFV) retrieved from all lineages (granulocytes, B cells, and T cells) at a single time point for each vector in ZJ41 and ZJ48. A threshold of 2,000 reads was applied to establish a master list of barcodes for each vector within each animal: to include a barcode on the master list, it must have contributed 2,000 reads at at least one time point. Once established on the master list, this barcode would be counted even at time points where it contributed less than 2,000 reads. The same barcode found contributing to more than one lineage was counted only once. (C) Cumulative number of unique barcodes retrieved for each vector (EF1-α, MSCV, and SFFV) from all three lineages combined (granulocytes, B cells, and T cells) over time in ZJ41 and ZJ48. (D) Simpson’s diversity index for all lineages combined (granulocytes, B cells, and T cells) combined in ZJ41 and ZJ48.
    Barcodes, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 4110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc pcr amplified
    iCLIP identifies the TIA1 and TIAL1 crosslink sites with nucleotide resolution. Autoradiogram of 32 P-labelled RNA crosslinked to TIA1 (A) or TIAL1 (B) in HeLa cells. Immunoprecipitation was performed with either anti-TIA1 or anti-TIAL1 antibody using lysate from UV-crosslinked HeLa cells, cells transfected with TIA1 or TIAL1, TIA1/TIAL1 KD cells, or non-crosslinked cells. High and low RNase concentrations were used and protein G beads were used as a control. The Western blots below the autoradiograms show the input lysate used for each immunoprecipitation. (C) TIA1 and TIAL1 crosslink to uridine tracts downstream of the alternative 5′ splice sites in the CLIP4 gene. The <t>cDNA</t> positions are colour-coded for three replicate TIA1 and TIAL1 experiments, and the random barcode (shown on the left) is used to identify unique iCLIP cDNAs (number in brackets indicates the number of corresponding <t>PCR</t> duplicates). Below, the bar graphs show the cDNA count (number of cDNAs at each crosslink site). Pre-mRNA sequence is shown below with crosslink nucleotides in red. The exon and intron positions of the two isoforms of CLIP4 mRNA are shown at the bottom.
    Pcr Amplified, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 2551 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc nextera xt dna library preparation kit
    NGS-library preparation using the homemade Tn5 constructs. (A) Workflow of Tn5 loading, cDNA tagmentation, and subsequent NGS library preparation for duplex index (Illumina i5/i7) full-length cDNA sequencing. Tn5 molecules are shown as gray hexamers. The double-stranded part of the linker oligonucleotide, the mosaic element, is shown in gray with a yellow circle depicting the phosphorylated 3′ end. The 5′ overhangs as templates for the i5 or i7 index adapter primers are shown in red or blue, respectively. cDNA is shown as two parallel black lines with a 3′ poly(A) tail. The synthesis of the 5′ overhang complementary strand (gap-filling step during PCR amplification) is depicted as a dotted arrow. i5 index adapter primer is shown in dark blue, while i7 index adapter primer is shown in orange. Fragments that are lost during library preparation are transparent. (B) Bioanalyzer traces of NGS libraries processed with different concentrations of the in-house-produced Tn5 R27S,E54K,L372P using only homemade or inexpensive commercially available reagents for tagmentation and subsequent PCR reaction. Following the tagmentation protocol presented here, fragmentation of the cDNA works best when using Tn5 R27S,E54K,L372P at a concentration of 30 ng/μl. (C) Heat scatter plot showing the correlation of read counts between libraries processed with either in-house-produced Tn5 E54K,L372P or Tn5 R27S,E54K,L372P . Data of three technical replicates per condition were pooled for this analysis. (D) Heat scatter plot showing the correlation of gene counts between libraries processed using either in-house-produced Tn5 R27S,E54K,L372P and the protocol presented here or the <t>Nextera</t> XT <t>DNA</t> library preparation kit following the manufacturer’s instructions. Data of three technical replicates per condition were pooled for this analysis. (E) Pairwise correlation of read counts between three technical replicates (samples processed from the same cDNA on the same day) when using homemade Tn5 R27S,E54K,L372P and the tagmentation protocol presented here. The Pearson correlation of r = 0.99 between all samples demonstrates high reproducibility of both the enzyme and the protocol. (F) Heat map analysis of gene counts in technical replicates processed from the same cDNA but on different days. The color code indicates the Pearson correlation between samples (see legend on the right side). NGS, next-generation sequencing; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate.
    Nextera Xt Dna Library Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 4388 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Phylogeny of 34 most abundant operational taxonomic units (OTUs) of ur e C (corresponding to the species level). ur e C reads obtained by amplicon sequencing on the Illumina MiSeq platform were clustered into species-level OTUs with ≥91% sequence identity, and a phylogenetic tree was constructed by the maximum likelihood method with the Jones-Taylor-Thornton model by means of the ur e C sequence of Canavalia ensiformis (M65260) as an outgroup. Branching points that support probability > 80% in the bootstrap analyses (based on 500 replicates) are shown as filled circles. The heatmap shows the relative abundance of species-level OTUs of ure C in soil samples 1 to 7. The scale bar represents 20% sequence divergence. Nucleotide sequence accession numbers are indicated in parentheses.

    Journal: Microbes and Environments

    Article Title: Ureolytic Prokaryotes in Soil: Community Abundance and Diversity

    doi: 10.1264/jsme2.ME17188

    Figure Lengend Snippet: Phylogeny of 34 most abundant operational taxonomic units (OTUs) of ur e C (corresponding to the species level). ur e C reads obtained by amplicon sequencing on the Illumina MiSeq platform were clustered into species-level OTUs with ≥91% sequence identity, and a phylogenetic tree was constructed by the maximum likelihood method with the Jones-Taylor-Thornton model by means of the ur e C sequence of Canavalia ensiformis (M65260) as an outgroup. Branching points that support probability > 80% in the bootstrap analyses (based on 500 replicates) are shown as filled circles. The heatmap shows the relative abundance of species-level OTUs of ure C in soil samples 1 to 7. The scale bar represents 20% sequence divergence. Nucleotide sequence accession numbers are indicated in parentheses.

    Article Snippet: PCR products were tagged with a sampleunique index and Illumina adapter sequences at their 5′ end (Nextera XT Index Kit v2; Illumina, San Diego, CA, USA) by PCR, and sequenced on the Illumina MiSeq platform in a 250-bp paired-end sequencing reaction with the v2 reagent kit (Illumina).

    Techniques: Amplification, Sequencing, Construct

    NGS results. A fragment covering the entire CRISPR target site was amplified (76 fish) prior to Illumina MiSeq sequencing. When reporting read counts with the inserted sequence, we distinguished between the following groups; (a) Perfect HDR (reads with a perfect match to the entire target sequence), and (b) Perfect FLAG + indels (reads with a correct insert sequence but mismatches/indels in the homology arms). We also reported reads with mismatches in the insert sequence (referred to as degenerated FLAG) and wild type reads (Supplementary Fig. 3 ). All the data are summarized in (c) . Read counts for each group are given in % of the total number of reads with at least 100 identical reads, for each sample. Individual samples are represented by black diamonds, and grouped for each of the different repair templates, at different concentrations (represented by grey bars). The error bars indicate the SEM of the mean for each group. Non-parametric statistics (Kruskall-Wallis) were performed to analyze the differences in HDR efficiencies between the different repair templates. Different lower-case letters indicate significant differences ( P

    Journal: Scientific Reports

    Article Title: Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon

    doi: 10.1038/s41598-019-57295-w

    Figure Lengend Snippet: NGS results. A fragment covering the entire CRISPR target site was amplified (76 fish) prior to Illumina MiSeq sequencing. When reporting read counts with the inserted sequence, we distinguished between the following groups; (a) Perfect HDR (reads with a perfect match to the entire target sequence), and (b) Perfect FLAG + indels (reads with a correct insert sequence but mismatches/indels in the homology arms). We also reported reads with mismatches in the insert sequence (referred to as degenerated FLAG) and wild type reads (Supplementary Fig. 3 ). All the data are summarized in (c) . Read counts for each group are given in % of the total number of reads with at least 100 identical reads, for each sample. Individual samples are represented by black diamonds, and grouped for each of the different repair templates, at different concentrations (represented by grey bars). The error bars indicate the SEM of the mean for each group. Non-parametric statistics (Kruskall-Wallis) were performed to analyze the differences in HDR efficiencies between the different repair templates. Different lower-case letters indicate significant differences ( P

    Article Snippet: In order to verify insertion of FLAG and to assess the level of mosaicism, a fragment covering the entire target site was amplified in selected samples (n = 76) with a two-step fusion PCR to prepare for sequencing by Illumina MiSeq, as described in .

    Techniques: Next-Generation Sequencing, CRISPR, Amplification, Fluorescence In Situ Hybridization, Sequencing

    PCR amplification of the mitochondrial (mt) minichromosomes of the Polyplax rat lice. (A) Lane 1: GeneRuler®100 bp DNA Ladder (Thermo Scientific). Lane 2: PCR amplicons generated with primer pair 57F-57R that spans the coding region of each mitochondrial (mt) minichromosome of Polyplax asiatica . Lane 4: PCR amplicons generated with primer pair 301F-301R that spans the coding region of each mt minichromosome of Polyplax spinulosa . (B) PCR verification of the mt minichromosomes of Po. asiatica . Lane 1 and 13: 500 bp DNA Ladder (TIANGEN). Lane 2–12: PCR amplicons from the 11 minichromosomes of Po. asiatica : atp8 - atp6 , trnE - cob - trnI , cox1 - trnL 2 ( taa ), trnD - trnY - cox2 - nad6 , trnR - nad4L - cox3 - trnA , trnS 1 ( tct )- trnS 2 ( tga )- nad1 - trnT - trnG - nad3 - trnW , trnQ - nad2 - trnN - trnP , trnK - nad4 - trnF , trnH - nad5 , rrnS - trnC , trnM - trnL 1 ( tag )- rrnL - trnV . (C) PCR verification of the mt minichromosomes of Po. spinulosa . Lane 1 and 13: 500 bp DNA Ladder (TIANGEN). Lane 2–12: PCR amplicons from the 11 minichromosomes of Po. spinulosa : atp8 - atp6 , trnE - cob - trnI , cox1 - trnL 1 ( tag ), trnT - trnD - trnY - cox2 - nad6 - trnA , trnR - nad4L - trnP - cox3 , nad1 - trnG - nad3 - trnW , trnQ - nad2 - trnN , trnK - nad4 , trnH - nad5 - trnF , trnS 1 ( tct )- trnS 2 ( tga )- rrnS - trnC , trnM - trnL 2 ( taa )- rrnL - trnV . Genes from which PCR primers were designed are in bold.

    Journal: BMC Genomics

    Article Title: Fragmented mitochondrial genomes of the rat lice, Polyplax asiatica and Polyplax spinulosa: intra-genus variation in fragmentation pattern and a possible link between the extent of fragmentation and the length of life cycle

    doi: 10.1186/1471-2164-15-44

    Figure Lengend Snippet: PCR amplification of the mitochondrial (mt) minichromosomes of the Polyplax rat lice. (A) Lane 1: GeneRuler®100 bp DNA Ladder (Thermo Scientific). Lane 2: PCR amplicons generated with primer pair 57F-57R that spans the coding region of each mitochondrial (mt) minichromosome of Polyplax asiatica . Lane 4: PCR amplicons generated with primer pair 301F-301R that spans the coding region of each mt minichromosome of Polyplax spinulosa . (B) PCR verification of the mt minichromosomes of Po. asiatica . Lane 1 and 13: 500 bp DNA Ladder (TIANGEN). Lane 2–12: PCR amplicons from the 11 minichromosomes of Po. asiatica : atp8 - atp6 , trnE - cob - trnI , cox1 - trnL 2 ( taa ), trnD - trnY - cox2 - nad6 , trnR - nad4L - cox3 - trnA , trnS 1 ( tct )- trnS 2 ( tga )- nad1 - trnT - trnG - nad3 - trnW , trnQ - nad2 - trnN - trnP , trnK - nad4 - trnF , trnH - nad5 , rrnS - trnC , trnM - trnL 1 ( tag )- rrnL - trnV . (C) PCR verification of the mt minichromosomes of Po. spinulosa . Lane 1 and 13: 500 bp DNA Ladder (TIANGEN). Lane 2–12: PCR amplicons from the 11 minichromosomes of Po. spinulosa : atp8 - atp6 , trnE - cob - trnI , cox1 - trnL 1 ( tag ), trnT - trnD - trnY - cox2 - nad6 - trnA , trnR - nad4L - trnP - cox3 , nad1 - trnG - nad3 - trnW , trnQ - nad2 - trnN , trnK - nad4 , trnH - nad5 - trnF , trnS 1 ( tct )- trnS 2 ( tga )- rrnS - trnC , trnM - trnL 2 ( taa )- rrnL - trnV . Genes from which PCR primers were designed are in bold.

    Article Snippet: These amplicons were sequenced with Illumina Hiseq 2000 platform at the BGI-HK.

    Techniques: Polymerase Chain Reaction, Amplification, Generated

    Illumina MiSeq sequencing analysis.

    Journal: Applied and Environmental Microbiology

    Article Title: Microbiota Dynamics Associated with Environmental Conditions and Potential Roles of Cellulolytic Communities in Traditional Chinese Cereal Starter Solid-State Fermentation

    doi: 10.1128/AEM.01325-15

    Figure Lengend Snippet: Illumina MiSeq sequencing analysis.

    Article Snippet: Consequently, the Illumina MiSeq sequencing provided high detection of microbial communities and was superior to the PCR-DGGE in detecting the rare communities.

    Techniques: Sequencing

    Illumina MiSeq sequencing analysis.

    Journal: Applied and Environmental Microbiology

    Article Title: Microbiota Dynamics Associated with Environmental Conditions and Potential Roles of Cellulolytic Communities in Traditional Chinese Cereal Starter Solid-State Fermentation

    doi: 10.1128/AEM.01325-15

    Figure Lengend Snippet: Illumina MiSeq sequencing analysis.

    Article Snippet: Consequently, the Illumina MiSeq sequencing provided high detection of microbial communities and was superior to the PCR-DGGE in detecting the rare communities.

    Techniques: Sequencing

    PCoA/PCA of microbial communities in samples. (a) Weighted UniFrac distance PCoA of bacterial communities in samples as obtained by Illumina MiSeq sequencing; (b) Bray-Curtis distance PCoA of fungal communities in samples as obtained by Illumina MiSeq

    Journal: Applied and Environmental Microbiology

    Article Title: Microbiota Dynamics Associated with Environmental Conditions and Potential Roles of Cellulolytic Communities in Traditional Chinese Cereal Starter Solid-State Fermentation

    doi: 10.1128/AEM.01325-15

    Figure Lengend Snippet: PCoA/PCA of microbial communities in samples. (a) Weighted UniFrac distance PCoA of bacterial communities in samples as obtained by Illumina MiSeq sequencing; (b) Bray-Curtis distance PCoA of fungal communities in samples as obtained by Illumina MiSeq

    Article Snippet: Consequently, the Illumina MiSeq sequencing provided high detection of microbial communities and was superior to the PCR-DGGE in detecting the rare communities.

    Techniques: Sequencing

    Procedural schematic. Endoscopic lavage samples were collected from the cecum and sigmoid colon of each subject. The microbial and metabolic components of each sample were analyzed using Illumina-HiSeq 2000 and ultra performance liquid chromatography (UPLC)-mass spectrometry (MS), respectively. The analytic pipeline thereafter is shown. See methods for additional details. OTU: operational taxonomic unit; PICRUSt, phylotypic investigation of communities by reconstruction of unobserved states; HUMAnN: HMP unified metabolic analysis network; PCA, Principal Component Analysis.

    Journal: Microbiome

    Article Title: Integrative analysis of the microbiome and metabolome of the human intestinal mucosal surface reveals exquisite inter-relationships

    doi: 10.1186/2049-2618-1-17

    Figure Lengend Snippet: Procedural schematic. Endoscopic lavage samples were collected from the cecum and sigmoid colon of each subject. The microbial and metabolic components of each sample were analyzed using Illumina-HiSeq 2000 and ultra performance liquid chromatography (UPLC)-mass spectrometry (MS), respectively. The analytic pipeline thereafter is shown. See methods for additional details. OTU: operational taxonomic unit; PICRUSt, phylotypic investigation of communities by reconstruction of unobserved states; HUMAnN: HMP unified metabolic analysis network; PCA, Principal Component Analysis.

    Article Snippet: DNA sequencing was performed using an Illumina HiSeq 2000 (Illumina, Inc., San Diego, CA, USA).

    Techniques: Liquid Chromatography, Mass Spectrometry

    Recombination frequency during simultaneous amplification of multiple distinct HIV-1 genomes by next generation sequencing. A mixture of 35 genetically distinct HIV-1 genomes was subjected to PCR amplification. The PCR was performed with different copies of templates (3.5×10 4 , 3.5×10 5 or 3.5×10 6 copies) using different thermal cycle numbers (30, 35, 40 and 45). The PCR products were sequenced using a two-direction 600 cycle reagent kit on MiSeq. The merged sequences from two overlapping reads of the same cluster were then aligned to the HIV-1 reference sequence. The frequencies of all 35 parental sequence and their recombinants were determined by linkage analysis of 139 informative sites in each amplicon sequence using Nautilus [36] .

    Journal: PLoS ONE

    Article Title: Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR

    doi: 10.1371/journal.pone.0106658

    Figure Lengend Snippet: Recombination frequency during simultaneous amplification of multiple distinct HIV-1 genomes by next generation sequencing. A mixture of 35 genetically distinct HIV-1 genomes was subjected to PCR amplification. The PCR was performed with different copies of templates (3.5×10 4 , 3.5×10 5 or 3.5×10 6 copies) using different thermal cycle numbers (30, 35, 40 and 45). The PCR products were sequenced using a two-direction 600 cycle reagent kit on MiSeq. The merged sequences from two overlapping reads of the same cluster were then aligned to the HIV-1 reference sequence. The frequencies of all 35 parental sequence and their recombinants were determined by linkage analysis of 139 informative sites in each amplicon sequence using Nautilus [36] .

    Article Snippet: The second round PCR products were purified to eliminate the primer dimer, quantified by qPCR on an ABI 7300 realtime PCR machine, and sequenced using a two-direction 600 cycle reagent kit on MiSeq (Illumina, San Diego, CA).

    Techniques: Amplification, Next-Generation Sequencing, Polymerase Chain Reaction, Sequencing

    Schematic representation of the paired-end library preparation using a two-step tailed PCR. The workflow is derived from a document ‘16S metagenomic sequencing library preparation: preparing 16S ribosomal gene amplicons for the Illumina MiSeq system’ distributed by Illumina (part no. 15044223 Rev. B) and the figure was drawn with reference to a website of the Genomics and Sequencing Center at the University of Rhode Island ( http://web.uri.edu/gsc/next-generation-sequencing/ ).

    Journal: Royal Society Open Science

    Article Title: MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species

    doi: 10.1098/rsos.150088

    Figure Lengend Snippet: Schematic representation of the paired-end library preparation using a two-step tailed PCR. The workflow is derived from a document ‘16S metagenomic sequencing library preparation: preparing 16S ribosomal gene amplicons for the Illumina MiSeq system’ distributed by Illumina (part no. 15044223 Rev. B) and the figure was drawn with reference to a website of the Genomics and Sequencing Center at the University of Rhode Island ( http://web.uri.edu/gsc/next-generation-sequencing/ ).

    Article Snippet: Massively parallel paired-end sequencing on the MiSeq platform (Illumina, San Diego, CA, USA) requires PCR amplicons to be flanked by: (i) primer-binding sites for sequencing; (ii) dual-index (i.e. barcode) sequences; and (iii) adapter sequences for binding to the flowcells of the MiSeq.

    Techniques: Polymerase Chain Reaction, Derivative Assay, Sequencing, Next-Generation Sequencing

    FASTQC results pre- and post-filtering with Cutadapt. FASTQC results are from a single sample from the original set of 96 HS samples prepared in 12-plex and sequenced on the Illumina HiSeq 2500 with 125bp reads.

    Journal: bioRxiv

    Article Title: Adapting genotyping-by-sequencing and variant calling for heterogeneous stock rats

    doi: 10.1101/523043

    Figure Lengend Snippet: FASTQC results pre- and post-filtering with Cutadapt. FASTQC results are from a single sample from the original set of 96 HS samples prepared in 12-plex and sequenced on the Illumina HiSeq 2500 with 125bp reads.

    Article Snippet: As a pilot, an initial 96 HS samples were sequenced, 12 samples per library, at Beckman Coulter Genomics (now GENEWIZ) on an Illumina HiSeq 2500 with v4 chemistry and 125bp single-end reads.

    Techniques:

    Overview of the sequencing assay used to characterize blood meal composition of individual mosquitoes. (i) A first PCR amplification is performed on DNA extracted from each mosquito targeting ~140 bp of the mammalian mt 16S rRNA (gray) using primers modified with a 5’-end tail complementary to the Illumina sequencing primers (red). (ii) A second PCR amplification incorporates the Illumina adaptors and a 6-nucleotide barcode unique to each mosquito at the ends of the individual blood meal PCR products. (iii) After pooling amplification products from 96 samples together, the PCR products are simultaneously sequenced on an Illumina MiSeq to (iv) generate paired-end reads (in grey) and barcode sequences (grey box). (v) Paired-end reads are then merged to provide error-corrected consensus sequence reads. The dotted black line indicates that the 6-nucleotide barcode corresponding to each read is known but is sequenced independently.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Unbiased Characterization of Anopheles Mosquito Blood Meals by Targeted High-Throughput Sequencing

    doi: 10.1371/journal.pntd.0004512

    Figure Lengend Snippet: Overview of the sequencing assay used to characterize blood meal composition of individual mosquitoes. (i) A first PCR amplification is performed on DNA extracted from each mosquito targeting ~140 bp of the mammalian mt 16S rRNA (gray) using primers modified with a 5’-end tail complementary to the Illumina sequencing primers (red). (ii) A second PCR amplification incorporates the Illumina adaptors and a 6-nucleotide barcode unique to each mosquito at the ends of the individual blood meal PCR products. (iii) After pooling amplification products from 96 samples together, the PCR products are simultaneously sequenced on an Illumina MiSeq to (iv) generate paired-end reads (in grey) and barcode sequences (grey box). (v) Paired-end reads are then merged to provide error-corrected consensus sequence reads. The dotted black line indicates that the 6-nucleotide barcode corresponding to each read is known but is sequenced independently.

    Article Snippet: We characterized the blood meal composition of a total of 442 female Anopheles by amplifying the mammalian mt 16S rRNA genes from DNA extracted from these mosquitoes, pooling the PCR products of 96 samples after individual barcoding, and simultaneously sequencing the samples on an Illumina MiSeq instrument ( ).

    Techniques: Sequencing, Polymerase Chain Reaction, Amplification, Modification

    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: Microbiome analysis of ticks using next generation sequencing by Illumina technology The V4 region of the 16S rRNA gene of each tick was amplified using previously described primers [ ].

    Techniques: Amplification

    PR8-induced IFN-Is alter the fecal microbiota composition, Analysis of fecal microbiota in WT and Ifnar1 -/- mice performed by MiSeq and 16S qPCR during influenza infection. A) Experimental model. Fecal samples were collected from mice on day 0 before infection and on day 9 after mock and PR8 infection. Mice were euthanized at 17 dpi. B, C) The fecal microbiota from WT and Ifnar1 -/- mice on day 0 before infection (n = 6 WT, n = 6 Ifnar1 -/- ), and on day 9 after mock (n = 3 WT, n = 3 Ifnar1 -/- ) and PR8 infection (n = 3 WT, n = 3 Ifnar1 -/- ) was analyzed by sequencing using the Illumina MiSeq system. Graphed is the average relative abundance of each bacterial phylum (B) and genus (C); the cut-off abundance level was set at 0.5%. D) Analysis of the fecal Enterobacteriaceae using 16S qPCR. Fecal samples were collected from mice on day 0 before infection (n = 10 WT, n = 8 Ifnar1 -/- ) and on day 9 after mock (n = 5 WT, n = 4 Ifnar1 -/- ) and PR8 infection (n = 5 WT, n = 4 Ifnar1 -/- ). Copy numbers of Enterobacteriaceae per μl of fecal microbial DNA is shown. Each dot represents one mouse, the geometric mean is indicated. P values were calculated by One-Way ANOVA (Bonferroni multiple comparison test). ***p

    Journal: PLoS Pathogens

    Article Title: Influenza Virus Affects Intestinal Microbiota and Secondary Salmonella Infection in the Gut through Type I Interferons

    doi: 10.1371/journal.ppat.1005572

    Figure Lengend Snippet: PR8-induced IFN-Is alter the fecal microbiota composition, Analysis of fecal microbiota in WT and Ifnar1 -/- mice performed by MiSeq and 16S qPCR during influenza infection. A) Experimental model. Fecal samples were collected from mice on day 0 before infection and on day 9 after mock and PR8 infection. Mice were euthanized at 17 dpi. B, C) The fecal microbiota from WT and Ifnar1 -/- mice on day 0 before infection (n = 6 WT, n = 6 Ifnar1 -/- ), and on day 9 after mock (n = 3 WT, n = 3 Ifnar1 -/- ) and PR8 infection (n = 3 WT, n = 3 Ifnar1 -/- ) was analyzed by sequencing using the Illumina MiSeq system. Graphed is the average relative abundance of each bacterial phylum (B) and genus (C); the cut-off abundance level was set at 0.5%. D) Analysis of the fecal Enterobacteriaceae using 16S qPCR. Fecal samples were collected from mice on day 0 before infection (n = 10 WT, n = 8 Ifnar1 -/- ) and on day 9 after mock (n = 5 WT, n = 4 Ifnar1 -/- ) and PR8 infection (n = 5 WT, n = 4 Ifnar1 -/- ). Copy numbers of Enterobacteriaceae per μl of fecal microbial DNA is shown. Each dot represents one mouse, the geometric mean is indicated. P values were calculated by One-Way ANOVA (Bonferroni multiple comparison test). ***p

    Article Snippet: Libraries were sequenced using an Illumina MiSeq system.

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Infection, Sequencing

    Targeted validation of promoter mutations a , Targeted sequencing validation of selected promoter mutations in 47 patients from the ExomePlus cohort with Illumina TruSeq Custom Amplicon panel (TSCA)-targeted sequencing technology. b , Validation rate of promoter mutations calculated as validated mutations over all sequenced and powered mutations. c , Median detection sensitivity at mutated sites for significantly mutated promoters. Each point indicates a single mutated position. d , PCR-MiSeq for the FOXA1 promoter locus for 126 patients with sufficient coverage for mutation calling from the original ExomePlus cohort. Three out of four mutations validated in experiment (green and red bars). PCR-MiSeq for 140 patients included but not covered in original ExomePlus experiment and 64 additional tumours yielded three novel mutations in each set (light and dark blue bars). No germline mutations at this site were detected in normal samples.

    Journal: Nature

    Article Title: Recurrent and functional regulatory mutations in breast cancer

    doi: 10.1038/nature22992

    Figure Lengend Snippet: Targeted validation of promoter mutations a , Targeted sequencing validation of selected promoter mutations in 47 patients from the ExomePlus cohort with Illumina TruSeq Custom Amplicon panel (TSCA)-targeted sequencing technology. b , Validation rate of promoter mutations calculated as validated mutations over all sequenced and powered mutations. c , Median detection sensitivity at mutated sites for significantly mutated promoters. Each point indicates a single mutated position. d , PCR-MiSeq for the FOXA1 promoter locus for 126 patients with sufficient coverage for mutation calling from the original ExomePlus cohort. Three out of four mutations validated in experiment (green and red bars). PCR-MiSeq for 140 patients included but not covered in original ExomePlus experiment and 64 additional tumours yielded three novel mutations in each set (light and dark blue bars). No germline mutations at this site were detected in normal samples.

    Article Snippet: The samples were hybridized with their custom oligonucleotide pool and then run through a series of steps consisting of washing, extension and ligation of the bound oligos, and PCR amplification, where Illumina custom i5 and i7 sequencing primers were added to the final product.

    Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Mutagenesis

    Hh infection induces IL-22 dependent dysbiosis (A) Quantification of Hh colonization density in the cecal content. n=9-11. (B) Chao and Shannon diversity indices from 16S rRNA sequencing data obtained from fecal pellets of uninfected mice and Hh infected mice treated with control or anti-IL-22 antibody as indicated. (C) Relative abundance of indicated taxa from groups described in B. (D) Relative abundance of indicated taxa as determined by taxa specific 16s rRNA qPCR from groups described in B.

    Journal: Mucosal immunology

    Article Title: Interleukin-22 drives nitric oxide-dependent DNA damage and dysplasia in a murine model of colitis-associated cancer

    doi: 10.1038/mi.2017.9

    Figure Lengend Snippet: Hh infection induces IL-22 dependent dysbiosis (A) Quantification of Hh colonization density in the cecal content. n=9-11. (B) Chao and Shannon diversity indices from 16S rRNA sequencing data obtained from fecal pellets of uninfected mice and Hh infected mice treated with control or anti-IL-22 antibody as indicated. (C) Relative abundance of indicated taxa from groups described in B. (D) Relative abundance of indicated taxa as determined by taxa specific 16s rRNA qPCR from groups described in B.

    Article Snippet: Amplicons were generated using oligonucleotide primers that target approximately 300 bp of the V4 variable region of the 16S rRNA gene (primers 515F and 806R) and also were barcoded and pooled to construct the sequencing library, followed by sequencing with an Illumina MiSeq instrument to generate paired-end 150×150 reads.

    Techniques: Infection, Sequencing, Mouse Assay, Real-time Polymerase Chain Reaction

    Overview of Experimental Methods (A) Schematic of the phenotypes studied. (B) Schematic of the PBAL method. Cells are lysed in 96-well plates to release genomic DNA (gDNA) and then subjected to bisulfite conversion that simultaneously converts and shears the gDNA into fragments. Random hexamers are then used to regenerate double-stranded DNA that is then end-repaired, a-tailed, and ligated with indexed adapters before low-cycle PCR amplification. Libraries are then pooled and sequenced on an Illumina sequencing platform. See also Figures S1 and S2 .

    Journal: Stem Cell Reports

    Article Title: High-Resolution Single-Cell DNA Methylation Measurements Reveal Epigenetically Distinct Hematopoietic Stem Cell Subpopulations

    doi: 10.1016/j.stemcr.2018.07.003

    Figure Lengend Snippet: Overview of Experimental Methods (A) Schematic of the phenotypes studied. (B) Schematic of the PBAL method. Cells are lysed in 96-well plates to release genomic DNA (gDNA) and then subjected to bisulfite conversion that simultaneously converts and shears the gDNA into fragments. Random hexamers are then used to regenerate double-stranded DNA that is then end-repaired, a-tailed, and ligated with indexed adapters before low-cycle PCR amplification. Libraries are then pooled and sequenced on an Illumina sequencing platform. See also Figures S1 and S2 .

    Article Snippet: The double-stranded DNA was then ligated with forked Illumina adapters ( ) and PCR amplified for eight cycles.

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing

    Relative expression of BRCA2 ▼3 and ∆3 transcripts in six c.68‐7T > A carriers and 12 normal controls by quantitative PCR. The boxplots (displaying low, Q1, median, Q3, and high values) show qPCR levels of ▼3 and ∆3 transcripts in carriers and controls. Values are normalized to GUSB mRNA and expressed as fold difference relative to pooled control cDNAs using the ∆ΔCq method (see Materials and Methods). The analysis shows in carriers a statistically significant increase of the relative level of ∆3 transcripts compared to controls (2.98 vs. 0.97; p

    Journal: Human Mutation

    Article Title: The BRCA2 c.68‐7T  >  A variant is not pathogenic: A model for clinical calibration of spliceogenicity, et al. The BRCA2 c.68‐7T  >  A variant is not pathogenic: a model for clinical calibration of spliceogenicity

    doi: 10.1002/humu.23411

    Figure Lengend Snippet: Relative expression of BRCA2 ▼3 and ∆3 transcripts in six c.68‐7T > A carriers and 12 normal controls by quantitative PCR. The boxplots (displaying low, Q1, median, Q3, and high values) show qPCR levels of ▼3 and ∆3 transcripts in carriers and controls. Values are normalized to GUSB mRNA and expressed as fold difference relative to pooled control cDNAs using the ∆ΔCq method (see Materials and Methods). The analysis shows in carriers a statistically significant increase of the relative level of ∆3 transcripts compared to controls (2.98 vs. 0.97; p

    Article Snippet: The qPCR analysis were performed on the Eco real‐time PCR system (Illumina) using SYBR® Green I dye chemistry (KAPA SYBR® FAST qPCR Kit, Kapa Biosystems).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Graphical summary of the SL-seq protocol. (A) cDNA generation with Superscript III and a primer that is partially random (7 nucleotides, grey), and partially fixed (yellow). Consequent degradation of the RNA strand with RNAse H, leaving a single stranded DNA molecule. (B) Second strand synthesis of SL-containing DNA molecules with Klenow fragment and a primer that is complementary with the SL (dark blue). (C + D) PCR for amplification and addition of adapter motives (red and purple) making the library compatible with the Nextera XT index kit (Illumina). (E + F) Final PCR adapter extension, indexing (orange and light blue) and amplification of the library fragments with the primers of the Nextera XT index kit. Dark Blue: SL/ complementary with SL, light grey: RNA, dark grey: DNA, green: poly-A tail. Other colors: primer and adapters sequences.

    Journal: Scientific Reports

    Article Title: Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids

    doi: 10.1038/s41598-017-03987-0

    Figure Lengend Snippet: Graphical summary of the SL-seq protocol. (A) cDNA generation with Superscript III and a primer that is partially random (7 nucleotides, grey), and partially fixed (yellow). Consequent degradation of the RNA strand with RNAse H, leaving a single stranded DNA molecule. (B) Second strand synthesis of SL-containing DNA molecules with Klenow fragment and a primer that is complementary with the SL (dark blue). (C + D) PCR for amplification and addition of adapter motives (red and purple) making the library compatible with the Nextera XT index kit (Illumina). (E + F) Final PCR adapter extension, indexing (orange and light blue) and amplification of the library fragments with the primers of the Nextera XT index kit. Dark Blue: SL/ complementary with SL, light grey: RNA, dark grey: DNA, green: poly-A tail. Other colors: primer and adapters sequences.

    Article Snippet: Addition of Illumina adapters, amplification and sample indexing Additional adapter sequences were added on 5′ and 3′ end of the library fragments to make them compatible with the Nextera XT Index Kit (Illumina).

    Techniques: Polymerase Chain Reaction, Amplification

    Outline of BAsE-Seq methodology. (a) The goal of library preparation is to attach unique barcodes to full-length HBV genomes, and then juxtapose the assigned barcode to random overlapping fragments of the viral genome. A unique barcode is first assigned to each HBV genome using PCR. The two barcode assignment primers contain HBV-specific sequences on their 3′ ends, universal sequences (green) on their 5′ ends, and one of the primers also contains a random barcode (blue). Subsequently, barcode-tagged genomes are clonally amplified by PCR using primers that anneal to Uni-A and Uni-B and that add a biotin label (Bio) to the barcode-proximal end. The barcode-distal end is digested with exonuclease to obtain a broad size distribution of nested deletion fragments. Barcode-containing fragments are purified using Dynabeads, and intramolecular ligation of these fragments yields a library of circular molecules in which different regions of each HBV genome are juxtaposed to its assigned barcode. The circularized molecules are used as a template for random fragmentation and adapter tagging following the Nextera protocol. During PCR enrichment, a set of primers is used to incorporate Illumina-specific paired-end adapters and enrich for barcode-tagged molecules during sequencing. (b) Bioinformatics workflow. Barcode-containing read pairs are used to obtain a 'bulk consensus' genome by iterative alignment of read pairs against a GenBank sequence. Aligned read pairs are de-multiplexed into individual genomes based on barcode identity. Consensus base calls are extracted to obtain 'individual consensus' genomes and SNVs are identified in each genome to construct haplotypes.

    Journal: Genome Biology

    Article Title: BAsE-Seq: a method for obtaining long viral haplotypes from short sequence reads

    doi: 10.1186/s13059-014-0517-9

    Figure Lengend Snippet: Outline of BAsE-Seq methodology. (a) The goal of library preparation is to attach unique barcodes to full-length HBV genomes, and then juxtapose the assigned barcode to random overlapping fragments of the viral genome. A unique barcode is first assigned to each HBV genome using PCR. The two barcode assignment primers contain HBV-specific sequences on their 3′ ends, universal sequences (green) on their 5′ ends, and one of the primers also contains a random barcode (blue). Subsequently, barcode-tagged genomes are clonally amplified by PCR using primers that anneal to Uni-A and Uni-B and that add a biotin label (Bio) to the barcode-proximal end. The barcode-distal end is digested with exonuclease to obtain a broad size distribution of nested deletion fragments. Barcode-containing fragments are purified using Dynabeads, and intramolecular ligation of these fragments yields a library of circular molecules in which different regions of each HBV genome are juxtaposed to its assigned barcode. The circularized molecules are used as a template for random fragmentation and adapter tagging following the Nextera protocol. During PCR enrichment, a set of primers is used to incorporate Illumina-specific paired-end adapters and enrich for barcode-tagged molecules during sequencing. (b) Bioinformatics workflow. Barcode-containing read pairs are used to obtain a 'bulk consensus' genome by iterative alignment of read pairs against a GenBank sequence. Aligned read pairs are de-multiplexed into individual genomes based on barcode identity. Consensus base calls are extracted to obtain 'individual consensus' genomes and SNVs are identified in each genome to construct haplotypes.

    Article Snippet: The circularized molecules were used as a template for random fragmentation and adapter tagging using the Nextera XT kit (Illumina, San Diego, CA, USA).

    Techniques: Polymerase Chain Reaction, Amplification, Purification, Ligation, Sequencing, Construct

    Experimental design. GFP reporter expression was driven by the EF-1α promoter and potentially modulated by a variable 8mer inserted into the human IQGAP1 3′UTR. The 5′UTR of the GFP reporter contains an intron. Expression of dsRed was used to control for transcriptional noise at the reporter locus, and was driven by the PGK promoter. The flippase recombination target (FRT) site allows this plasmid to undergo site-specific recombination in HEK293-TRex-FLP cells, such that only cells that integrate this construct at the intended locus via the FRT site gain hygromycin resistance. After integration, cells with normal transcriptional activity at the reporter locus (as determined by dsRed levels), and that are potentially undergoing differential post-transcriptional regulation (as determined by GFP levels), were isolated via fluorescence activated cell sorting (FACS). From FACS isolated sub-populations, the portion of the 3′UTR containing the variable 8mer was PCR amplified, thereby adding Illumina adapter sequences, and allowing 8mers in each sorted population to be identified and quantified by Illumina sequencing

    Journal: BMC Genomics

    Article Title: High-throughput discovery of post-transcriptional cis-regulatory elements

    doi: 10.1186/s12864-016-2479-7

    Figure Lengend Snippet: Experimental design. GFP reporter expression was driven by the EF-1α promoter and potentially modulated by a variable 8mer inserted into the human IQGAP1 3′UTR. The 5′UTR of the GFP reporter contains an intron. Expression of dsRed was used to control for transcriptional noise at the reporter locus, and was driven by the PGK promoter. The flippase recombination target (FRT) site allows this plasmid to undergo site-specific recombination in HEK293-TRex-FLP cells, such that only cells that integrate this construct at the intended locus via the FRT site gain hygromycin resistance. After integration, cells with normal transcriptional activity at the reporter locus (as determined by dsRed levels), and that are potentially undergoing differential post-transcriptional regulation (as determined by GFP levels), were isolated via fluorescence activated cell sorting (FACS). From FACS isolated sub-populations, the portion of the 3′UTR containing the variable 8mer was PCR amplified, thereby adding Illumina adapter sequences, and allowing 8mers in each sorted population to be identified and quantified by Illumina sequencing

    Article Snippet: We then used PCR to add barcodes and Illumina sequencing adapters to the region surrounding the variable 8-nt region; all oligonucleotides are listed in Additional file .

    Techniques: Expressing, Plasmid Preparation, Construct, Activity Assay, Isolation, Fluorescence, FACS, Polymerase Chain Reaction, Amplification, Sequencing

    Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

    Journal: BMC Genomics

    Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing

    doi: 10.1186/1471-2164-14-355

    Figure Lengend Snippet: Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

    Article Snippet: Cycling parameters were as follows: initial denaturation of 94°C/2 min followed by 30 cycles of 98°C/10 sec, 60°C/15 sec and 68°C/10 min. DNA libraries of these PCR products were prepared by a transposase-mediated library preparation method with the Nextera DNA Sample Preparation Kit (Illumina, San Diego, CA, USA) allowing reduced amount of DNA (50 ng) and time for the library construction (90 min).

    Techniques: Selection, Agarose Gel Electrophoresis, Sample Prep, Concentration Assay, Bridge PCR, Amplification

    Expression levels of 11 PCGs in the nymph (A), pupa (B) and adult (C) of MED whiteflies. Real-Time PCR was used to detect genes expression levels. The x-axis shows the different PCGs, and the y-axis represents the mRNA expression level of different developing stages compared to each control (β-actin).

    Journal: BMC Genomics

    Article Title: The characteristics and expression profiles of the mitochondrial genome for the Mediterranean species of the Bemisia tabaci complex

    doi: 10.1186/1471-2164-14-401

    Figure Lengend Snippet: Expression levels of 11 PCGs in the nymph (A), pupa (B) and adult (C) of MED whiteflies. Real-Time PCR was used to detect genes expression levels. The x-axis shows the different PCGs, and the y-axis represents the mRNA expression level of different developing stages compared to each control (β-actin).

    Article Snippet: However, PCR primers can be designed according to the MED Illumina reads mapped to the New World mitogenome with nearly 100% confidence, therefore improve the probability of success (see Additional file ).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Vector Contributions and Clonal Diversity of EF1-α, MSCV, and SFFV Barcoded Vectors (A) The percentage contribution of each vector (EF1-α, MSCV, and SFFV) to total transduced hematopoiesis in granulocytes, B cells, and T cells purified from the blood of animals ZJ41 and ZJ48 over time post-transplantation. The read number of each vector’s library ID in a sample over the total read numbers for all library IDs in the sample ×100 is plotted. (B) Number of unique barcodes for each vector (EF1-α, MSCV, and SFFV) retrieved from all lineages (granulocytes, B cells, and T cells) at a single time point for each vector in ZJ41 and ZJ48. A threshold of 2,000 reads was applied to establish a master list of barcodes for each vector within each animal: to include a barcode on the master list, it must have contributed 2,000 reads at at least one time point. Once established on the master list, this barcode would be counted even at time points where it contributed less than 2,000 reads. The same barcode found contributing to more than one lineage was counted only once. (C) Cumulative number of unique barcodes retrieved for each vector (EF1-α, MSCV, and SFFV) from all three lineages combined (granulocytes, B cells, and T cells) over time in ZJ41 and ZJ48. (D) Simpson’s diversity index for all lineages combined (granulocytes, B cells, and T cells) combined in ZJ41 and ZJ48.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Barcoding of Macaque Hematopoietic Stem and Progenitor Cells: A Robust Platform to Assess Vector Genotoxicity

    doi: 10.1016/j.omtm.2018.10.009

    Figure Lengend Snippet: Vector Contributions and Clonal Diversity of EF1-α, MSCV, and SFFV Barcoded Vectors (A) The percentage contribution of each vector (EF1-α, MSCV, and SFFV) to total transduced hematopoiesis in granulocytes, B cells, and T cells purified from the blood of animals ZJ41 and ZJ48 over time post-transplantation. The read number of each vector’s library ID in a sample over the total read numbers for all library IDs in the sample ×100 is plotted. (B) Number of unique barcodes for each vector (EF1-α, MSCV, and SFFV) retrieved from all lineages (granulocytes, B cells, and T cells) at a single time point for each vector in ZJ41 and ZJ48. A threshold of 2,000 reads was applied to establish a master list of barcodes for each vector within each animal: to include a barcode on the master list, it must have contributed 2,000 reads at at least one time point. Once established on the master list, this barcode would be counted even at time points where it contributed less than 2,000 reads. The same barcode found contributing to more than one lineage was counted only once. (C) Cumulative number of unique barcodes retrieved for each vector (EF1-α, MSCV, and SFFV) from all three lineages combined (granulocytes, B cells, and T cells) over time in ZJ41 and ZJ48. (D) Simpson’s diversity index for all lineages combined (granulocytes, B cells, and T cells) combined in ZJ41 and ZJ48.

    Article Snippet: To assess clonal patterns in vivo at time points from 1 month through 33 and 38 months post-transplantation, blood was obtained, and granulocytes, T cells, and B cells were lineage purified by flow cytrometry, followed by DNA extraction, low-cycle PCR with primers bracketing the library IDs and barcodes, high-throughput Illumina sequencing, and data processing to retrieve and quantitate the read fraction for each individual barcode linked to one of the three vector-associated library IDs.

    Techniques: Plasmid Preparation, Purification, Transplantation Assay

    iCLIP identifies the TIA1 and TIAL1 crosslink sites with nucleotide resolution. Autoradiogram of 32 P-labelled RNA crosslinked to TIA1 (A) or TIAL1 (B) in HeLa cells. Immunoprecipitation was performed with either anti-TIA1 or anti-TIAL1 antibody using lysate from UV-crosslinked HeLa cells, cells transfected with TIA1 or TIAL1, TIA1/TIAL1 KD cells, or non-crosslinked cells. High and low RNase concentrations were used and protein G beads were used as a control. The Western blots below the autoradiograms show the input lysate used for each immunoprecipitation. (C) TIA1 and TIAL1 crosslink to uridine tracts downstream of the alternative 5′ splice sites in the CLIP4 gene. The cDNA positions are colour-coded for three replicate TIA1 and TIAL1 experiments, and the random barcode (shown on the left) is used to identify unique iCLIP cDNAs (number in brackets indicates the number of corresponding PCR duplicates). Below, the bar graphs show the cDNA count (number of cDNAs at each crosslink site). Pre-mRNA sequence is shown below with crosslink nucleotides in red. The exon and intron positions of the two isoforms of CLIP4 mRNA are shown at the bottom.

    Journal: PLoS Biology

    Article Title: iCLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions

    doi: 10.1371/journal.pbio.1000530

    Figure Lengend Snippet: iCLIP identifies the TIA1 and TIAL1 crosslink sites with nucleotide resolution. Autoradiogram of 32 P-labelled RNA crosslinked to TIA1 (A) or TIAL1 (B) in HeLa cells. Immunoprecipitation was performed with either anti-TIA1 or anti-TIAL1 antibody using lysate from UV-crosslinked HeLa cells, cells transfected with TIA1 or TIAL1, TIA1/TIAL1 KD cells, or non-crosslinked cells. High and low RNase concentrations were used and protein G beads were used as a control. The Western blots below the autoradiograms show the input lysate used for each immunoprecipitation. (C) TIA1 and TIAL1 crosslink to uridine tracts downstream of the alternative 5′ splice sites in the CLIP4 gene. The cDNA positions are colour-coded for three replicate TIA1 and TIAL1 experiments, and the random barcode (shown on the left) is used to identify unique iCLIP cDNAs (number in brackets indicates the number of corresponding PCR duplicates). Below, the bar graphs show the cDNA count (number of cDNAs at each crosslink site). Pre-mRNA sequence is shown below with crosslink nucleotides in red. The exon and intron positions of the two isoforms of CLIP4 mRNA are shown at the bottom.

    Article Snippet: Linearised cDNA was then PCR-amplified using primers complementary to the adapter regions and subjected to high-throughput sequencing using Illumina GA2.

    Techniques: Immunoprecipitation, Transfection, Western Blot, Polymerase Chain Reaction, Sequencing

    NGS-library preparation using the homemade Tn5 constructs. (A) Workflow of Tn5 loading, cDNA tagmentation, and subsequent NGS library preparation for duplex index (Illumina i5/i7) full-length cDNA sequencing. Tn5 molecules are shown as gray hexamers. The double-stranded part of the linker oligonucleotide, the mosaic element, is shown in gray with a yellow circle depicting the phosphorylated 3′ end. The 5′ overhangs as templates for the i5 or i7 index adapter primers are shown in red or blue, respectively. cDNA is shown as two parallel black lines with a 3′ poly(A) tail. The synthesis of the 5′ overhang complementary strand (gap-filling step during PCR amplification) is depicted as a dotted arrow. i5 index adapter primer is shown in dark blue, while i7 index adapter primer is shown in orange. Fragments that are lost during library preparation are transparent. (B) Bioanalyzer traces of NGS libraries processed with different concentrations of the in-house-produced Tn5 R27S,E54K,L372P using only homemade or inexpensive commercially available reagents for tagmentation and subsequent PCR reaction. Following the tagmentation protocol presented here, fragmentation of the cDNA works best when using Tn5 R27S,E54K,L372P at a concentration of 30 ng/μl. (C) Heat scatter plot showing the correlation of read counts between libraries processed with either in-house-produced Tn5 E54K,L372P or Tn5 R27S,E54K,L372P . Data of three technical replicates per condition were pooled for this analysis. (D) Heat scatter plot showing the correlation of gene counts between libraries processed using either in-house-produced Tn5 R27S,E54K,L372P and the protocol presented here or the Nextera XT DNA library preparation kit following the manufacturer’s instructions. Data of three technical replicates per condition were pooled for this analysis. (E) Pairwise correlation of read counts between three technical replicates (samples processed from the same cDNA on the same day) when using homemade Tn5 R27S,E54K,L372P and the tagmentation protocol presented here. The Pearson correlation of r = 0.99 between all samples demonstrates high reproducibility of both the enzyme and the protocol. (F) Heat map analysis of gene counts in technical replicates processed from the same cDNA but on different days. The color code indicates the Pearson correlation between samples (see legend on the right side). NGS, next-generation sequencing; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate.

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol

    doi: 10.1534/g3.117.300257

    Figure Lengend Snippet: NGS-library preparation using the homemade Tn5 constructs. (A) Workflow of Tn5 loading, cDNA tagmentation, and subsequent NGS library preparation for duplex index (Illumina i5/i7) full-length cDNA sequencing. Tn5 molecules are shown as gray hexamers. The double-stranded part of the linker oligonucleotide, the mosaic element, is shown in gray with a yellow circle depicting the phosphorylated 3′ end. The 5′ overhangs as templates for the i5 or i7 index adapter primers are shown in red or blue, respectively. cDNA is shown as two parallel black lines with a 3′ poly(A) tail. The synthesis of the 5′ overhang complementary strand (gap-filling step during PCR amplification) is depicted as a dotted arrow. i5 index adapter primer is shown in dark blue, while i7 index adapter primer is shown in orange. Fragments that are lost during library preparation are transparent. (B) Bioanalyzer traces of NGS libraries processed with different concentrations of the in-house-produced Tn5 R27S,E54K,L372P using only homemade or inexpensive commercially available reagents for tagmentation and subsequent PCR reaction. Following the tagmentation protocol presented here, fragmentation of the cDNA works best when using Tn5 R27S,E54K,L372P at a concentration of 30 ng/μl. (C) Heat scatter plot showing the correlation of read counts between libraries processed with either in-house-produced Tn5 E54K,L372P or Tn5 R27S,E54K,L372P . Data of three technical replicates per condition were pooled for this analysis. (D) Heat scatter plot showing the correlation of gene counts between libraries processed using either in-house-produced Tn5 R27S,E54K,L372P and the protocol presented here or the Nextera XT DNA library preparation kit following the manufacturer’s instructions. Data of three technical replicates per condition were pooled for this analysis. (E) Pairwise correlation of read counts between three technical replicates (samples processed from the same cDNA on the same day) when using homemade Tn5 R27S,E54K,L372P and the tagmentation protocol presented here. The Pearson correlation of r = 0.99 between all samples demonstrates high reproducibility of both the enzyme and the protocol. (F) Heat map analysis of gene counts in technical replicates processed from the same cDNA but on different days. The color code indicates the Pearson correlation between samples (see legend on the right side). NGS, next-generation sequencing; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate.

    Article Snippet: In a first attempt to test the activity of Tn5E54K,L372P and Tn5R27S,E54K,L372P , we performed tagmentation reactions with reagents supplied in the Illumina Nextera XT DNA library preparation kit following the manufacturer’s instructions, but substituting the Nextera ATM enzyme with one of the two loaded in-house-produced Tn5 versions.

    Techniques: Next-Generation Sequencing, Construct, Sequencing, Polymerase Chain Reaction, Amplification, Produced, Concentration Assay