hiseq 2500 Illumina Inc Search Results


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  • 99
    Illumina Inc illumina hiseq 2500
    Experimental scheme. ( A ) After differentiation, the cell cultures were dissociated to single-cell suspensions and loaded onto a Fluidigm C1 Single-Cell Auto Prep Array for mRNA-Seq. On the arrays, the cells were lysed, and reverse transcription of the mRNA and PCR amplification of the cDNA were performed using the C1 Single-Cell Auto Prep System (Fluidigm). Libraries were then prepared using the Nextera XT DNA Library Prep Kit and mosquito HTS liquid handler (TTP). For the final PCR reactions, we used a Bio-Rad 384 Thermal Cycler. Libraries were pooled and sequenced on an <t>Illumina</t> HiSeq 2500. ( B ) WA09 human embryonic stem cells were differentiated in vitro to the pancreatic lineage. Cells from stages 1 and 2 were collected and analyzed using the procedures outlined in A . Two independent cells from stage 1 (cell A and cell B) and two cells from stage 2 (cell C and cell D) were selected and yielded with similar cDNA concentrations (mean [SD] concentration = 0.38 [0.04] ng/µL). For library preparation, we tested 2-µL, 4-µL, and 8-µL final volume reactions, with four replicates per reaction volume.
    Illumina Hiseq 2500, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 56070 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Illumina Inc cdna libraries
    Imprinting of FWA is observed in the endosperm of mature seed. ( A ) Confocal images showing FWA-GFP localization in the mature endosperm of the reciprocally crossed F1 seeds. The images were made using confocal microscopy of female pFWA::dFWA-GFP x male pDD65::mtKaede (upper panels) and female pDD65::mtKaede x male pFWA::dFWA-GFP (lower panels) endosperms. Endosperm tissue autofluorescence and FWA-GFP fluorescence signals were captured through the band-pass filter for GFP (left panels). The endosperm cells were visualized by DIC optic (middle panels), and both images were merged (right panels). Although broad background fluorescence were visible in the cytosol in both directions of cross, some cells showed nuclear-localized green fluorescence that is only detectable in the female pFWA::dFWA-GFP cross. pDD65::mtKaede signals distributed with a speckle-like pattern (that probably corresponds to the distribution of mitochondria) in the cytosolic space of all cells. The low signal intensity might be due to the low expression level of FWA in mature seeds, especially in the Col-0 background ( Figure 2—source data 3 ). Bar = 20 μm. ( B ) RT-PCR detecting the FWA-GFP transgenic mRNA in the mature endosperm of F1 seeds. <t>RNA</t> extracted from endosperm dissected 36 hr after imbibition was analyzed by RT-PCR. FWA- GFP <t>cDNA</t> was detected in the endosperm of pFWA::dFWA-GFP x pDD65::mtKaede and pFWA::dFWA-GFP x Col F1 seeds but not in that of pDD65::mtKaede x pFWA::dFWA-GFP and Col x pFWA::dFWA-GFP F1 seeds. ACT11 is used as a positive control. DOI: http://dx.doi.org/10.7554/eLife.19573.018
    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
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    92
    Illumina Inc hiseq 2500 sequencing system
    Illustrated representation of the optimized barcoded cDNA library preparation procedure. Total RNA from formalin-fixed paraffin-embedded (FFPE) specimens ( n = 18), are ligated with 3′ adapters ( n = 18), purified on a 15% PAGE gel (using size markers (SM) RNA oligonucleotides) and excised after SYBR ® gold staining of the gel. The combined purified products are ligated with a 5′ adapter, run on a 12% PAGE gel, excised and purified. The dual-adapter ligated small-RNA products are reverse transcribed, subjected to a pilot PCR for identification of adequate amplification cycle. A large-scale PCR is performed, and the products are separated on a 2% agarose gel, where the upper band (cDNA library) is purified and subjected to sequencing on an Illumina <t>HiSeq-2500</t> sequencer.
    Hiseq 2500 Sequencing System, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 1631 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc rna seq library
    KDM2B regulates apoptotic machinery at transcriptional level in GBM cells. ( a ) Ingenuity pathway analysis of <t>RNA-seq</t> results. ( b ) List of the top 10 canonical pathways that were most significantly upregulated or downregulated upon KDM2B loss. ( c ) Custom list of genes displaying top deregulated apoptosis-related genes along with transcript level of KDM2B ( n =2 biological replicates, average number of reads of shKDM2B cells were normalized to <t>shControl</t> cells). ( d ) qRT-PCR validation of HRK gene expression levels of shKDM2B cells. Expression levels were normalized to shControl. ( e ) Western blots showing HRK upregulation in shKDM2B cells at the protein level ( f ) qRT-PCR quantification of HRK mRNA level of HRK knockdown cells. Expression levels were normalized to shControl. ( g ) Cell viability analysis upon TRAIL treatment of shControl and shHRK cells transduced with either shControl or shKDM2B. Data were normalized to untreated cells of each group. (*,** and *** denotes P
    Rna Seq Library, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 2453 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc rna sequencing
    KDM2B regulates apoptotic machinery at transcriptional level in GBM cells. ( a ) Ingenuity pathway analysis of <t>RNA-seq</t> results. ( b ) List of the top 10 canonical pathways that were most significantly upregulated or downregulated upon KDM2B loss. ( c ) Custom list of genes displaying top deregulated apoptosis-related genes along with transcript level of KDM2B ( n =2 biological replicates, average number of reads of shKDM2B cells were normalized to <t>shControl</t> cells). ( d ) qRT-PCR validation of HRK gene expression levels of shKDM2B cells. Expression levels were normalized to shControl. ( e ) Western blots showing HRK upregulation in shKDM2B cells at the protein level ( f ) qRT-PCR quantification of HRK mRNA level of HRK knockdown cells. Expression levels were normalized to shControl. ( g ) Cell viability analysis upon TRAIL treatment of shControl and shHRK cells transduced with either shControl or shKDM2B. Data were normalized to untreated cells of each group. (*,** and *** denotes P
    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
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    94
    Illumina Inc cbot cluster generation system
    KDM2B regulates apoptotic machinery at transcriptional level in GBM cells. ( a ) Ingenuity pathway analysis of <t>RNA-seq</t> results. ( b ) List of the top 10 canonical pathways that were most significantly upregulated or downregulated upon KDM2B loss. ( c ) Custom list of genes displaying top deregulated apoptosis-related genes along with transcript level of KDM2B ( n =2 biological replicates, average number of reads of shKDM2B cells were normalized to <t>shControl</t> cells). ( d ) qRT-PCR validation of HRK gene expression levels of shKDM2B cells. Expression levels were normalized to shControl. ( e ) Western blots showing HRK upregulation in shKDM2B cells at the protein level ( f ) qRT-PCR quantification of HRK mRNA level of HRK knockdown cells. Expression levels were normalized to shControl. ( g ) Cell viability analysis upon TRAIL treatment of shControl and shHRK cells transduced with either shControl or shKDM2B. Data were normalized to untreated cells of each group. (*,** and *** denotes P
    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
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    93
    Illumina Inc genomic dna
    Acute exposure to Pb modifies the <t>DNA</t> hydroxymethylation (5hmC) profile of <t>hESCs</t>
    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
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    95
    Illumina Inc paired end sequencing
    Acute exposure to Pb modifies the <t>DNA</t> hydroxymethylation (5hmC) profile of <t>hESCs</t>
    Paired End Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 95/100, based on 9460 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Illumina Inc hiseq 2000 2500 platform
    Study design Fibroblast cells were isolated from adult C57BL/6J, SPRET/EiJ, and the F1 hybrid mice and cultured. PolyA RNAs prepared from each cell line were sequenced on an Illumina HiSeq 2000/2500 platform.
    Hiseq 2000 2500 Platform, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 441 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Illumina Inc srna libraries
    Small RNAs complementary to the PiGPB1 gene. Distribution of small RNA reads homologous to the PiGPBI gene in transgenic hp-PiGPB1 and wild-type potato plants at 24, 48, and 72 hpi based on <t>Illumina</t> sequencing.
    Srna Libraries, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 827 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc illumina cbot
    Small RNAs complementary to the PiGPB1 gene. Distribution of small RNA reads homologous to the PiGPBI gene in transgenic hp-PiGPB1 and wild-type potato plants at 24, 48, and 72 hpi based on <t>Illumina</t> sequencing.
    Illumina Cbot, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 4815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc truseq stranded mrna library prep kit
    Small RNAs complementary to the PiGPB1 gene. Distribution of small RNA reads homologous to the PiGPBI gene in transgenic hp-PiGPB1 and wild-type potato plants at 24, 48, and 72 hpi based on <t>Illumina</t> sequencing.
    Truseq Stranded Mrna Library Prep Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 2678 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Experimental scheme. ( A ) After differentiation, the cell cultures were dissociated to single-cell suspensions and loaded onto a Fluidigm C1 Single-Cell Auto Prep Array for mRNA-Seq. On the arrays, the cells were lysed, and reverse transcription of the mRNA and PCR amplification of the cDNA were performed using the C1 Single-Cell Auto Prep System (Fluidigm). Libraries were then prepared using the Nextera XT DNA Library Prep Kit and mosquito HTS liquid handler (TTP). For the final PCR reactions, we used a Bio-Rad 384 Thermal Cycler. Libraries were pooled and sequenced on an Illumina HiSeq 2500. ( B ) WA09 human embryonic stem cells were differentiated in vitro to the pancreatic lineage. Cells from stages 1 and 2 were collected and analyzed using the procedures outlined in A . Two independent cells from stage 1 (cell A and cell B) and two cells from stage 2 (cell C and cell D) were selected and yielded with similar cDNA concentrations (mean [SD] concentration = 0.38 [0.04] ng/µL). For library preparation, we tested 2-µL, 4-µL, and 8-µL final volume reactions, with four replicates per reaction volume.

    Journal: Jala (Charlottesville, Va.)

    Article Title: Miniaturization Technologies for Efficient Single-Cell Library Preparation for Next-Generation Sequencing

    doi: 10.1177/2211068216630741

    Figure Lengend Snippet: Experimental scheme. ( A ) After differentiation, the cell cultures were dissociated to single-cell suspensions and loaded onto a Fluidigm C1 Single-Cell Auto Prep Array for mRNA-Seq. On the arrays, the cells were lysed, and reverse transcription of the mRNA and PCR amplification of the cDNA were performed using the C1 Single-Cell Auto Prep System (Fluidigm). Libraries were then prepared using the Nextera XT DNA Library Prep Kit and mosquito HTS liquid handler (TTP). For the final PCR reactions, we used a Bio-Rad 384 Thermal Cycler. Libraries were pooled and sequenced on an Illumina HiSeq 2500. ( B ) WA09 human embryonic stem cells were differentiated in vitro to the pancreatic lineage. Cells from stages 1 and 2 were collected and analyzed using the procedures outlined in A . Two independent cells from stage 1 (cell A and cell B) and two cells from stage 2 (cell C and cell D) were selected and yielded with similar cDNA concentrations (mean [SD] concentration = 0.38 [0.04] ng/µL). For library preparation, we tested 2-µL, 4-µL, and 8-µL final volume reactions, with four replicates per reaction volume.

    Article Snippet: Sequencing The 48 pooled libraries were sequenced on one lane of an Illumina HiSeq 2500 at an average total read depth of 5.6 million reads per sample (ST3).

    Techniques: Polymerase Chain Reaction, Amplification, In Vitro, Concentration Assay

    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

    (a) Hierarchical cluster analysis for genes included in non-fluorescent liposomes. Ten non-fluorescent liposomes that did not show β-galactoside hydrolysis activity were isolated, and the genes included in these liposomes were determined by multiplex next-generation sequencing using HiSeq 2500. In the hierarchical cluster analysis for these genes, there was no commonality among the genes included in each liposome, which indicated a random pattern. Each row represents a separate gene, each column represents a separate liposome and genes found in each liposome are shown in red. (b) Hierarchical cluster analysis for the genes included in fluorescent liposomes. Hierarchical cluster analysis revealed that  LacZ  was a clear cluster that was included in all fluorescent liposomes and with no other common factors.

    Journal: Scientific Reports

    Article Title: Integrating reductive and synthetic approaches in biology using man-made cell-like compartments

    doi: 10.1038/srep04722

    Figure Lengend Snippet: (a) Hierarchical cluster analysis for genes included in non-fluorescent liposomes. Ten non-fluorescent liposomes that did not show β-galactoside hydrolysis activity were isolated, and the genes included in these liposomes were determined by multiplex next-generation sequencing using HiSeq 2500. In the hierarchical cluster analysis for these genes, there was no commonality among the genes included in each liposome, which indicated a random pattern. Each row represents a separate gene, each column represents a separate liposome and genes found in each liposome are shown in red. (b) Hierarchical cluster analysis for the genes included in fluorescent liposomes. Hierarchical cluster analysis revealed that LacZ was a clear cluster that was included in all fluorescent liposomes and with no other common factors.

    Article Snippet: Next, the liposome-derived DNA fragments and the E. coli ORF library were pre-treated for HiSeq 2500 according to the Nextera XT DNA preparation kit protocol (Illumina).

    Techniques: Activity Assay, Isolation, Multiplex Assay, Next-Generation Sequencing

    Box plots of read depths at concordant/discordant SNP loci among the NGSs and Omni 2.5-8. A) Box plots of the NGS read depth at the SNP loci showing concordance/discordance between the NGSs and Omni 2.5-8. The box indicates the first and third quartiles and the lines indicate the highest and lowest value that is within 1.5 x inter-quartile range. Outliers were omitted. The graph on the left indicates the distribution of read depths at HiSeq2500 SNP loci and the graph on the right indicates the distribution of read depths at Ion Proton and Omni 2.5-8 SNP loci. B) Box plots of the NGS read depths at the SNP loci showing concordance/discordance among the three platforms. The box indicates the first and third quartiles and the lines indicate the highest and lowest value that is within 1.5 x inter-quartile range. Outliers were omitted. The three columns (shaded gray rectangle) from right indicate the distribution of read depths of the discordant SNPs between the NGSs and Omni 2.5-8. The read depth at SNP loci that showed Ion Proton discordance against HiSeq 2500 and Omni 2.5-8 was significantly different. The read depths at SNP loci for SNPs that both HiSeq 2500 and Ion Proton failed to call are shown in the gray box.

    Journal: BMC Genomics

    Article Title: Validation of multiple single nucleotide variation calls by additional exome analysis with a semiconductor sequencer to supplement data of whole-genome sequencing of a human population

    doi: 10.1186/1471-2164-15-673

    Figure Lengend Snippet: Box plots of read depths at concordant/discordant SNP loci among the NGSs and Omni 2.5-8. A) Box plots of the NGS read depth at the SNP loci showing concordance/discordance between the NGSs and Omni 2.5-8. The box indicates the first and third quartiles and the lines indicate the highest and lowest value that is within 1.5 x inter-quartile range. Outliers were omitted. The graph on the left indicates the distribution of read depths at HiSeq2500 SNP loci and the graph on the right indicates the distribution of read depths at Ion Proton and Omni 2.5-8 SNP loci. B) Box plots of the NGS read depths at the SNP loci showing concordance/discordance among the three platforms. The box indicates the first and third quartiles and the lines indicate the highest and lowest value that is within 1.5 x inter-quartile range. Outliers were omitted. The three columns (shaded gray rectangle) from right indicate the distribution of read depths of the discordant SNPs between the NGSs and Omni 2.5-8. The read depth at SNP loci that showed Ion Proton discordance against HiSeq 2500 and Omni 2.5-8 was significantly different. The read depths at SNP loci for SNPs that both HiSeq 2500 and Ion Proton failed to call are shown in the gray box.

    Article Snippet: Ten microliters of 2 nM libraries were denatured with an equal volume of 0.1 N sodium hydroxide; 1.5–2.0 pM of the denatured library was then used for on-board cluster generation on a HiSeq 2500 system (Illumina) with a TruSeq Rapid PE Cluster Kit (Illumina).

    Techniques: Next-Generation Sequencing

    Measuring technical variation using DNA ladders. a Density distribution of k-mer counts for each cn unit ( n = 570 k-mers per unit; 1 cn = red, 2 cn = yellow, 4 cn = green, and 8 cn = blue) in a simulated and experimental libraries (prepared with KAPA HyperPlus PCR-free and sequenced on Illumina HiSeq X Ten), with the mean, standard deviation and coefficient of variation indicated. For the experimental library, the n = 14 independent synthetic ladders where examined over 5 independent experiments with comparable results (see HiSeq X Ten/PCR-free libraries in Supplementary Figs. 5 , 6 , and 7a ). b Modeling the impact of common NGS technical variables such as error rate, library depth, and library complexity on the regression slope of the synthetic DNA ladder. c The panel illustrates the variability associated with cn units across a range of libraries generated with different sequencing technologies (e.g., ONT Nanopore MinION, ONT Nanopore PromethION, Illumina HiSeq X Ten, Illumina NextSeq 500, and Illumina HiSeq 2500 instruments) or prepared with alternative protocols (e.g., KAPA, KAPA HyperPlus PCR-free/PCR-based and Nextera XT kits, or target enrichment by oligonucleotide hybridization). The y axis indicates k-mer counts, whilst the x axis indicates the position across the sub-sequence length (600 nt). The mean k-mer count (opaque line) and standard deviation (transparent line) are determined across all n = 14 independent synthetic ladders in all the sequencing experiments.

    Journal: Nature Communications

    Article Title: A universal and independent synthetic DNA ladder for the quantitative measurement of genomic features

    doi: 10.1038/s41467-020-17445-5

    Figure Lengend Snippet: Measuring technical variation using DNA ladders. a Density distribution of k-mer counts for each cn unit ( n = 570 k-mers per unit; 1 cn = red, 2 cn = yellow, 4 cn = green, and 8 cn = blue) in a simulated and experimental libraries (prepared with KAPA HyperPlus PCR-free and sequenced on Illumina HiSeq X Ten), with the mean, standard deviation and coefficient of variation indicated. For the experimental library, the n = 14 independent synthetic ladders where examined over 5 independent experiments with comparable results (see HiSeq X Ten/PCR-free libraries in Supplementary Figs. 5 , 6 , and 7a ). b Modeling the impact of common NGS technical variables such as error rate, library depth, and library complexity on the regression slope of the synthetic DNA ladder. c The panel illustrates the variability associated with cn units across a range of libraries generated with different sequencing technologies (e.g., ONT Nanopore MinION, ONT Nanopore PromethION, Illumina HiSeq X Ten, Illumina NextSeq 500, and Illumina HiSeq 2500 instruments) or prepared with alternative protocols (e.g., KAPA, KAPA HyperPlus PCR-free/PCR-based and Nextera XT kits, or target enrichment by oligonucleotide hybridization). The y axis indicates k-mer counts, whilst the x axis indicates the position across the sub-sequence length (600 nt). The mean k-mer count (opaque line) and standard deviation (transparent line) are determined across all n = 14 independent synthetic ladders in all the sequencing experiments.

    Article Snippet: They were then sequenced on a HiSeq 2500 instrument (Illumina®) producing paired reads of 125 nt at the Kinghorn Centre for Clinical Genomics, Sydney, Australia.

    Techniques: Polymerase Chain Reaction, Standard Deviation, Next-Generation Sequencing, Generated, Sequencing, Hybridization

    Performance comparisons of BBT against FACS, BWA and BT2. Receiver operator characteristic curves of BBT and FACS using simulated 100 bp SE reads from Homo sapiens mixed with ( A ) E.coli and ( B ) Mus musculus filtered against an H.sapiens Bloom filter using a k -mer size of 25 bp; ( C ) CPU time benchmark comparing BT2 (for a range of built-in settings), BWA (using aln and mem settings), FACS and BBT, on one lane of human 2 × 150 bp PE Illumina HiSeq 2500 reads

    Journal: Bioinformatics

    Article Title: BioBloom tools: fast, accurate and memory-efficient host species sequence screening using bloom filters

    doi: 10.1093/bioinformatics/btu558

    Figure Lengend Snippet: Performance comparisons of BBT against FACS, BWA and BT2. Receiver operator characteristic curves of BBT and FACS using simulated 100 bp SE reads from Homo sapiens mixed with ( A ) E.coli and ( B ) Mus musculus filtered against an H.sapiens Bloom filter using a k -mer size of 25 bp; ( C ) CPU time benchmark comparing BT2 (for a range of built-in settings), BWA (using aln and mem settings), FACS and BBT, on one lane of human 2 × 150 bp PE Illumina HiSeq 2500 reads

    Article Snippet: 3.2 Benchmarking on experimental data We used a single lane of 2 × 150 bp PE human DNA reads ( https://basespace.illumina.com/run/716717/2x150-HiSeq-2500-demo-NA12878 ) generated with an Illumina HiSeq 2500 sequencer to benchmark computational performance.

    Techniques: FACS

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

    Journal: mSystems

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

    doi: 10.1128/mSystems.00029-19

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

    Article Snippet: In summary, to demonstrate the comparability of sequence data sets produced via different methods, 16S rRNA gene V3-V4 region sequence data sets were generated from low-biomass vaginal samples from women using both 1-step and 2-step PCR library construction methods and the Illumina HiSeq and MiSeq sequencing platforms.

    Techniques: Amplification, Polymerase Chain Reaction

    Work flow used to identify somatic variants in genomic DNA extracted from human lens epithelial cells. Sequencing libraries were enriched for genes of the WUCaMP2 panel ( Table 2 ) by targeted hybridization capture. The resulting library was sequenced on an Illumina HiSeq 2500 platform to obtain paired-end reads. Sequencing results were filtered at several levels. The reads were then aligned to a human reference genome (hg19), and PCR duplicates were removed. Variants were called using SAMtools and VarScan2 software. Finally, selected variants were inspected manually using the IGV visualization tool.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Somatic Variants in the Human Lens Epithelium: A Preliminary Assessment

    doi: 10.1167/iovs.16-19726

    Figure Lengend Snippet: Work flow used to identify somatic variants in genomic DNA extracted from human lens epithelial cells. Sequencing libraries were enriched for genes of the WUCaMP2 panel ( Table 2 ) by targeted hybridization capture. The resulting library was sequenced on an Illumina HiSeq 2500 platform to obtain paired-end reads. Sequencing results were filtered at several levels. The reads were then aligned to a human reference genome (hg19), and PCR duplicates were removed. Variants were called using SAMtools and VarScan2 software. Finally, selected variants were inspected manually using the IGV visualization tool.

    Article Snippet: Sequencing and Base Calling Multiplex sequencing of the DNA libraries was performed on a HiSeq 2500 sequencing platform (Illumina Inc., San Diego, CA, USA) to obtain paired end 101-bp reads.

    Techniques: Flow Cytometry, Sequencing, Hybridization, Polymerase Chain Reaction, Software

    Reads passing filter vs. cluster density on Illumina MiSeq and HiSeq instruments. Each data point represents a run (flowcell). Shaded areas denote supported ranges of cluster densities and expected output for different chemistries/kits as specified by Illumina. The colour gradient indicates the total percentage of bases reaching a quality score of 30 or higher per run. Trend lines are generated by generalised additive model fits using a cubic penalised regression spline. M = millions

    Journal: BMC Genomics

    Article Title: 2.7 million samples genotyped for HLA by next generation sequencing: lessons learned

    doi: 10.1186/s12864-017-3575-z

    Figure Lengend Snippet: Reads passing filter vs. cluster density on Illumina MiSeq and HiSeq instruments. Each data point represents a run (flowcell). Shaded areas denote supported ranges of cluster densities and expected output for different chemistries/kits as specified by Illumina. The colour gradient indicates the total percentage of bases reaching a quality score of 30 or higher per run. Trend lines are generated by generalised additive model fits using a cubic penalised regression spline. M = millions

    Article Snippet: Here, we report on our experiences in genotyping the HLA, CCR5, ABO, RHD and KIR genes using a direct amplicon sequencing approach on Illumina MiSeq and HiSeq 2500 instruments.

    Techniques: Generated

    Imprinting of FWA is observed in the endosperm of mature seed. ( A ) Confocal images showing FWA-GFP localization in the mature endosperm of the reciprocally crossed F1 seeds. The images were made using confocal microscopy of female pFWA::dFWA-GFP x male pDD65::mtKaede (upper panels) and female pDD65::mtKaede x male pFWA::dFWA-GFP (lower panels) endosperms. Endosperm tissue autofluorescence and FWA-GFP fluorescence signals were captured through the band-pass filter for GFP (left panels). The endosperm cells were visualized by DIC optic (middle panels), and both images were merged (right panels). Although broad background fluorescence were visible in the cytosol in both directions of cross, some cells showed nuclear-localized green fluorescence that is only detectable in the female pFWA::dFWA-GFP cross. pDD65::mtKaede signals distributed with a speckle-like pattern (that probably corresponds to the distribution of mitochondria) in the cytosolic space of all cells. The low signal intensity might be due to the low expression level of FWA in mature seeds, especially in the Col-0 background ( Figure 2—source data 3 ). Bar = 20 μm. ( B ) RT-PCR detecting the FWA-GFP transgenic mRNA in the mature endosperm of F1 seeds. RNA extracted from endosperm dissected 36 hr after imbibition was analyzed by RT-PCR. FWA- GFP cDNA was detected in the endosperm of pFWA::dFWA-GFP x pDD65::mtKaede and pFWA::dFWA-GFP x Col F1 seeds but not in that of pDD65::mtKaede x pFWA::dFWA-GFP and Col x pFWA::dFWA-GFP F1 seeds. ACT11 is used as a positive control. DOI: http://dx.doi.org/10.7554/eLife.19573.018

    Journal: eLife

    Article Title: Dormancy-specific imprinting underlies maternal inheritance of seed dormancy in Arabidopsis thaliana

    doi: 10.7554/eLife.19573

    Figure Lengend Snippet: Imprinting of FWA is observed in the endosperm of mature seed. ( A ) Confocal images showing FWA-GFP localization in the mature endosperm of the reciprocally crossed F1 seeds. The images were made using confocal microscopy of female pFWA::dFWA-GFP x male pDD65::mtKaede (upper panels) and female pDD65::mtKaede x male pFWA::dFWA-GFP (lower panels) endosperms. Endosperm tissue autofluorescence and FWA-GFP fluorescence signals were captured through the band-pass filter for GFP (left panels). The endosperm cells were visualized by DIC optic (middle panels), and both images were merged (right panels). Although broad background fluorescence were visible in the cytosol in both directions of cross, some cells showed nuclear-localized green fluorescence that is only detectable in the female pFWA::dFWA-GFP cross. pDD65::mtKaede signals distributed with a speckle-like pattern (that probably corresponds to the distribution of mitochondria) in the cytosolic space of all cells. The low signal intensity might be due to the low expression level of FWA in mature seeds, especially in the Col-0 background ( Figure 2—source data 3 ). Bar = 20 μm. ( B ) RT-PCR detecting the FWA-GFP transgenic mRNA in the mature endosperm of F1 seeds. RNA extracted from endosperm dissected 36 hr after imbibition was analyzed by RT-PCR. FWA- GFP cDNA was detected in the endosperm of pFWA::dFWA-GFP x pDD65::mtKaede and pFWA::dFWA-GFP x Col F1 seeds but not in that of pDD65::mtKaede x pFWA::dFWA-GFP and Col x pFWA::dFWA-GFP F1 seeds. ACT11 is used as a positive control. DOI: http://dx.doi.org/10.7554/eLife.19573.018

    Article Snippet: For partially dissected endosperm and fully dissected endosperm samples, the cDNA libraries were prepared from 200 ng total RNA using a TruSeq mRNA Library Prep Kit (Illumina, Switzerland).

    Techniques: Confocal Microscopy, Fluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Positive Control

    Illustrated representation of the optimized barcoded cDNA library preparation procedure. Total RNA from formalin-fixed paraffin-embedded (FFPE) specimens ( n = 18), are ligated with 3′ adapters ( n = 18), purified on a 15% PAGE gel (using size markers (SM) RNA oligonucleotides) and excised after SYBR ® gold staining of the gel. The combined purified products are ligated with a 5′ adapter, run on a 12% PAGE gel, excised and purified. The dual-adapter ligated small-RNA products are reverse transcribed, subjected to a pilot PCR for identification of adequate amplification cycle. A large-scale PCR is performed, and the products are separated on a 2% agarose gel, where the upper band (cDNA library) is purified and subjected to sequencing on an Illumina HiSeq-2500 sequencer.

    Journal: International Journal of Molecular Sciences

    Article Title: Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens

    doi: 10.3390/ijms18030627

    Figure Lengend Snippet: Illustrated representation of the optimized barcoded cDNA library preparation procedure. Total RNA from formalin-fixed paraffin-embedded (FFPE) specimens ( n = 18), are ligated with 3′ adapters ( n = 18), purified on a 15% PAGE gel (using size markers (SM) RNA oligonucleotides) and excised after SYBR ® gold staining of the gel. The combined purified products are ligated with a 5′ adapter, run on a 12% PAGE gel, excised and purified. The dual-adapter ligated small-RNA products are reverse transcribed, subjected to a pilot PCR for identification of adequate amplification cycle. A large-scale PCR is performed, and the products are separated on a 2% agarose gel, where the upper band (cDNA library) is purified and subjected to sequencing on an Illumina HiSeq-2500 sequencer.

    Article Snippet: The cDNA libraries were then sequenced (single-read 50 cycles) on a HiSeq 2500 Sequencing System (SY-401-2501, Illumina, San Diego, CA, USA).

    Techniques: cDNA Library Assay, Formalin-fixed Paraffin-Embedded, Purification, Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Sequencing

    KDM2B regulates apoptotic machinery at transcriptional level in GBM cells. ( a ) Ingenuity pathway analysis of RNA-seq results. ( b ) List of the top 10 canonical pathways that were most significantly upregulated or downregulated upon KDM2B loss. ( c ) Custom list of genes displaying top deregulated apoptosis-related genes along with transcript level of KDM2B ( n =2 biological replicates, average number of reads of shKDM2B cells were normalized to shControl cells). ( d ) qRT-PCR validation of HRK gene expression levels of shKDM2B cells. Expression levels were normalized to shControl. ( e ) Western blots showing HRK upregulation in shKDM2B cells at the protein level ( f ) qRT-PCR quantification of HRK mRNA level of HRK knockdown cells. Expression levels were normalized to shControl. ( g ) Cell viability analysis upon TRAIL treatment of shControl and shHRK cells transduced with either shControl or shKDM2B. Data were normalized to untreated cells of each group. (*,** and *** denotes P

    Journal: Cell Death & Disease

    Article Title: KDM2B, an H3K36-specific demethylase, regulates apoptotic response of GBM cells to TRAIL

    doi: 10.1038/cddis.2017.288

    Figure Lengend Snippet: KDM2B regulates apoptotic machinery at transcriptional level in GBM cells. ( a ) Ingenuity pathway analysis of RNA-seq results. ( b ) List of the top 10 canonical pathways that were most significantly upregulated or downregulated upon KDM2B loss. ( c ) Custom list of genes displaying top deregulated apoptosis-related genes along with transcript level of KDM2B ( n =2 biological replicates, average number of reads of shKDM2B cells were normalized to shControl cells). ( d ) qRT-PCR validation of HRK gene expression levels of shKDM2B cells. Expression levels were normalized to shControl. ( e ) Western blots showing HRK upregulation in shKDM2B cells at the protein level ( f ) qRT-PCR quantification of HRK mRNA level of HRK knockdown cells. Expression levels were normalized to shControl. ( g ) Cell viability analysis upon TRAIL treatment of shControl and shHRK cells transduced with either shControl or shKDM2B. Data were normalized to untreated cells of each group. (*,** and *** denotes P

    Article Snippet: RNA-seq and analysis Total RNAs of shControl and shKDM2B cells were isolated, RNA-seq library for each sample was prepared based on protocols on Illumina HiSeq 2500 to generate 50 bp single-end reads.

    Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Western Blot, Transduction

    lincRNA-IBIN ( CR44404 ) expression is strongly induced by Gram-positive and Gram-negative bacteria and its expression is regulated by the Toll and Imd pathways and a functional BAP complex. A) In a whole transcriptome analysis, 28 genes were more than 18-fold upregulated after a M . luteus infection. The highest upregulation in infected flies was found in a long non-coding RNA gene, CR44404 ( lincRNA-IBIN ). B) 16 upregulated lncRNA genes have more than a threefold expression change in response to a M . luteus ). C) lincRNA-IBIN expression is induced in Drosophila adults within two hours of an infection by M . luteus or E . cloacae . For fold-induction values, expression values in uninfected samples were set to 1. D) M . luteus -induced lincRNA-IBIN expression is dependent on the Toll pathway (the Toll pathway adaptor protein MyD88) function. E) E . cloacae- induced lincRNA-IBIN expression is dependent on the Imd pathway (Relish) function. In D and E , for fold-induction values, expression values in uninfected w , MyD88 IR samples were set to 1 F) lincRNA-IBIN expression is induced in Drosophila adults upon silencing of the Drosophila inhibitor of κB factor cactus . G) lincRNA-IBIN expression is induced in Drosophila larvae with the constitutively active form of the Toll receptor, Toll 10b . In F and G , for fold-induction values, expression values in uninfected/untreated w samples were set to 1. H) lincRNA-IBIN expression is also modestly induced by the ubiquitous overexpression of Imd with daughterless-GAL4 ( da > Imd ) in Drosophila larvae. For fold-induction values, the expression value of w , da > was set to 1. I-J) Both M . luteus and E . cloacae -induced lincRNA-IBIN expression is dependent on the functional chromatin remodeling BAP complex. In I and J , for fold-induction values, expression values in uninfected w , osa IR samples were set to 1.

    Journal: PLoS Pathogens

    Article Title: Immune-inducible non-coding RNA molecule lincRNA-IBIN connects immunity and metabolism in Drosophila melanogaster

    doi: 10.1371/journal.ppat.1007504

    Figure Lengend Snippet: lincRNA-IBIN ( CR44404 ) expression is strongly induced by Gram-positive and Gram-negative bacteria and its expression is regulated by the Toll and Imd pathways and a functional BAP complex. A) In a whole transcriptome analysis, 28 genes were more than 18-fold upregulated after a M . luteus infection. The highest upregulation in infected flies was found in a long non-coding RNA gene, CR44404 ( lincRNA-IBIN ). B) 16 upregulated lncRNA genes have more than a threefold expression change in response to a M . luteus ). C) lincRNA-IBIN expression is induced in Drosophila adults within two hours of an infection by M . luteus or E . cloacae . For fold-induction values, expression values in uninfected samples were set to 1. D) M . luteus -induced lincRNA-IBIN expression is dependent on the Toll pathway (the Toll pathway adaptor protein MyD88) function. E) E . cloacae- induced lincRNA-IBIN expression is dependent on the Imd pathway (Relish) function. In D and E , for fold-induction values, expression values in uninfected w , MyD88 IR samples were set to 1 F) lincRNA-IBIN expression is induced in Drosophila adults upon silencing of the Drosophila inhibitor of κB factor cactus . G) lincRNA-IBIN expression is induced in Drosophila larvae with the constitutively active form of the Toll receptor, Toll 10b . In F and G , for fold-induction values, expression values in uninfected/untreated w samples were set to 1. H) lincRNA-IBIN expression is also modestly induced by the ubiquitous overexpression of Imd with daughterless-GAL4 ( da > Imd ) in Drosophila larvae. For fold-induction values, the expression value of w , da > was set to 1. I-J) Both M . luteus and E . cloacae -induced lincRNA-IBIN expression is dependent on the functional chromatin remodeling BAP complex. In I and J , for fold-induction values, expression values in uninfected w , osa IR samples were set to 1.

    Article Snippet: For the whole transcriptome analyses, the preparation of the RNA libraries and Illumina HiSeq 2500 sequencing were carried out in the Finnish Microarray and Sequencing Centre (Turku, Finland), and the transcriptome data analyses were carried out at The Bioinformatics Unit at the Turku Centre for Biotechnology and Biocenter Finland.

    Techniques: Expressing, Radial Immuno Diffusion, Functional Assay, Infection, Over Expression

    Acute exposure to Pb modifies the DNA hydroxymethylation (5hmC) profile of hESCs

    Journal: Epigenetics

    Article Title: Lead exposure induces changes in 5-hydroxymethylcytosine clusters in CpG islands in human embryonic stem cells and umbilical cord blood

    doi: 10.1080/15592294.2015.1050172

    Figure Lengend Snippet: Acute exposure to Pb modifies the DNA hydroxymethylation (5hmC) profile of hESCs

    Article Snippet: Briefly, the whole genomic DNA extracted from control and Pb-treated hESCs, was digested with PvuRts1I and sequenced using 50 bps paired end sequencing reads in Illumina™ HiSeq 2500.

    Techniques:

    Study design Fibroblast cells were isolated from adult C57BL/6J, SPRET/EiJ, and the F1 hybrid mice and cultured. PolyA RNAs prepared from each cell line were sequenced on an Illumina HiSeq 2000/2500 platform.

    Journal: Molecular Systems Biology

    Article Title: Predominant contribution of cis-regulatory divergence in the evolution of mouse alternative splicing

    doi: 10.15252/msb.20145970

    Figure Lengend Snippet: Study design Fibroblast cells were isolated from adult C57BL/6J, SPRET/EiJ, and the F1 hybrid mice and cultured. PolyA RNAs prepared from each cell line were sequenced on an Illumina HiSeq 2000/2500 platform.

    Article Snippet: The libraries were sequenced in 2 × 100nt + 7 manner on HiSeq 2000/2500 platform (Illumina).

    Techniques: Isolation, Mouse Assay, Cell Culture

    Small RNAs complementary to the PiGPB1 gene. Distribution of small RNA reads homologous to the PiGPBI gene in transgenic hp-PiGPB1 and wild-type potato plants at 24, 48, and 72 hpi based on Illumina sequencing.

    Journal: Journal of Experimental Botany

    Article Title: Plant-mediated gene silencing restricts growth of the potato late blight pathogen Phytophthora infestans

    doi: 10.1093/jxb/erv094

    Figure Lengend Snippet: Small RNAs complementary to the PiGPB1 gene. Distribution of small RNA reads homologous to the PiGPBI gene in transgenic hp-PiGPB1 and wild-type potato plants at 24, 48, and 72 hpi based on Illumina sequencing.

    Article Snippet: Eight sRNA libraries were generated using the Illumina small RNA sample preparation kit and sequenced using Illumina HiSeq 2500 at SciLifeLab, Stockholm, Sweden.

    Techniques: Transgenic Assay, Sequencing