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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 789 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Recurrent and functional regulatory mutations in breast cancer"

Article Title: Recurrent and functional regulatory mutations in breast cancer

Journal: Nature

doi: 10.1038/nature22992

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.
Figure Legend 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.

Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction, Mutagenesis

2) Product Images from "Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients"

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2017.09.007

Morphological and Phenotypic Characterization of Freshly Isolated Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids (A–C) Phase-contrast microscope revealed the morphology of the freshly isolated human pachytene spermatocytes (A), spermatogonia (B), and round spermatids (C) of OA patients. (D–F) DIC microscope showed the morphological characteristics of the freshly isolated human pachytene spermatocytes (D), spermatogonia (E), and round spermatids (F) of OA patients. Scale bars, 20 μm (A–C) and 5 μm (D–F). (G) RT-PCR revealed the transcripts of GPR125 , RET , GFRA1 , THY1 , UCHL1 , MAGEA4 , and PLZF in the fleshly isolated spermatogonia, the expression of SYCP3 and SYCP1 in pachytene spermatocytes, and mRNA of TNP1 , TNP2 , PRM1 , PRM2 , and ACR in round spermatids. RNA without RT (RT-) but with PCR of GAPDH primers was utilized as negative controls, and GAPDH served as loading controls of total RNA.
Figure Legend Snippet: Morphological and Phenotypic Characterization of Freshly Isolated Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids (A–C) Phase-contrast microscope revealed the morphology of the freshly isolated human pachytene spermatocytes (A), spermatogonia (B), and round spermatids (C) of OA patients. (D–F) DIC microscope showed the morphological characteristics of the freshly isolated human pachytene spermatocytes (D), spermatogonia (E), and round spermatids (F) of OA patients. Scale bars, 20 μm (A–C) and 5 μm (D–F). (G) RT-PCR revealed the transcripts of GPR125 , RET , GFRA1 , THY1 , UCHL1 , MAGEA4 , and PLZF in the fleshly isolated spermatogonia, the expression of SYCP3 and SYCP1 in pachytene spermatocytes, and mRNA of TNP1 , TNP2 , PRM1 , PRM2 , and ACR in round spermatids. RNA without RT (RT-) but with PCR of GAPDH primers was utilized as negative controls, and GAPDH served as loading controls of total RNA.

Techniques Used: Isolation, Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction

3) Product Images from "RESA identifies mRNA regulatory sequences with high resolution"

Article Title: RESA identifies mRNA regulatory sequences with high resolution

Journal: Nature methods

doi: 10.1038/nmeth.4121

RESA identifies sequences that promote mRNA destabilization (a) Wild-type sequence coverage depletion over time (top) reveals two destabilization elements in the gdpd5a 3′UTR. 430LNA inhibits destabilization of only the miR-430 target (bottom). (b) Regulated regions are identified by searching for a ≥ 1.33 fold depletion in late versus early stages, within a ≥65-nt region with ≥100X sequencing coverage. (c) Biplot of RESA-predicted destabilization regions, separated by mode of regulation. (d) Wild-type coverage depletion around a canonical miR-430 6-mer site ( tbc1d25 ) and non-canonical seed mismatch site with 3′ compensatory pairing ( sgk3 ) (e) GFP reporter analysis of RESA-predicted regulatory element with different regulatory behaviors. GFP mRNA levels are assayed at early (2hpf; yellow circles) and late (8hpf; blue squares) stage by qRT-PCR and normalized to a control dsRed mRNA. Plot shows mean +/− SEM; **, P
Figure Legend Snippet: RESA identifies sequences that promote mRNA destabilization (a) Wild-type sequence coverage depletion over time (top) reveals two destabilization elements in the gdpd5a 3′UTR. 430LNA inhibits destabilization of only the miR-430 target (bottom). (b) Regulated regions are identified by searching for a ≥ 1.33 fold depletion in late versus early stages, within a ≥65-nt region with ≥100X sequencing coverage. (c) Biplot of RESA-predicted destabilization regions, separated by mode of regulation. (d) Wild-type coverage depletion around a canonical miR-430 6-mer site ( tbc1d25 ) and non-canonical seed mismatch site with 3′ compensatory pairing ( sgk3 ) (e) GFP reporter analysis of RESA-predicted regulatory element with different regulatory behaviors. GFP mRNA levels are assayed at early (2hpf; yellow circles) and late (8hpf; blue squares) stage by qRT-PCR and normalized to a control dsRed mRNA. Plot shows mean +/− SEM; **, P

Techniques Used: Sequencing, Quantitative RT-PCR

4) Product Images from "Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain"

Article Title: Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain

Journal: BMC Genomics

doi: 10.1186/1471-2164-12-518

Reproducibility of AEI assays ( GNB1L ) . (a) [Left] Comparison of the results of independent measurements of GNB1L AEI. (Each assay was performed independently, from the isolation of brain RNA through DNA sequencing.); [Right] Regression analysis. (b) Histogram of the distribution of r 2 values obtained in regression analysis of independent AEI assays for 40 candidate genes for which replica assays were carried out. (All data are shown). (c) [Left] Comparison of the results of independent measurements of GNB1L AEI using PCR/DNA sequencing-based (top) and PCR/SNaPShot ® -based (bottom) assays. [Right] Regression analysis. [Note: the Illumina-2 AEI ratios in (a) were matched with those obtained in the Illumina-1 AEI assay, which contained fewer samples. The full set of data from the Illumina-2 assay is shown in (c).]
Figure Legend Snippet: Reproducibility of AEI assays ( GNB1L ) . (a) [Left] Comparison of the results of independent measurements of GNB1L AEI. (Each assay was performed independently, from the isolation of brain RNA through DNA sequencing.); [Right] Regression analysis. (b) Histogram of the distribution of r 2 values obtained in regression analysis of independent AEI assays for 40 candidate genes for which replica assays were carried out. (All data are shown). (c) [Left] Comparison of the results of independent measurements of GNB1L AEI using PCR/DNA sequencing-based (top) and PCR/SNaPShot ® -based (bottom) assays. [Right] Regression analysis. [Note: the Illumina-2 AEI ratios in (a) were matched with those obtained in the Illumina-1 AEI assay, which contained fewer samples. The full set of data from the Illumina-2 assay is shown in (c).]

Techniques Used: Isolation, DNA Sequencing, Polymerase Chain Reaction

5) Product Images from "Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing"

Article Title: Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr598

Targeted bisulfite sequencing. ( a ) Illustrated are the steps involved in the preparation of biotinylated RNA capture probes (top left and right), whole-genome fragment input library (top middle) and hybrid selection-enriched output library (middle left and right). The captured DNA was treated with sodium bisulfite, amplified by PCR and sequenced using an Illumina GAIIx sequencer. ( b ) Examples of targeted bisulfite sequencing using solution hybrid selection. The tracks shown from the top to bottom are: sequences of the capture probes; the DNA methylation level at each CpG site derived from the bisulfite-sequencing reads; the sequencing depth at each CpG site; RefSeq genes; and annotated CGIs in the UCSC genome browser. Red and green colors indicate methylated and unmethylated-CpG sites, respectively. MCF7-1 and MCF7-2 are technical replicates. For each sample, two tracks (methylation level and read depth) are shown. ( c ). Distribution of normalized abundance for the captured targets shared among all samples. The x -axis is the normalized abundance of each captured target, which is calculated by dividing the counts of the target by the average counts of all targets. The y -axis is the fraction of probes with coverage equal to or greater than the normalized coverage.
Figure Legend Snippet: Targeted bisulfite sequencing. ( a ) Illustrated are the steps involved in the preparation of biotinylated RNA capture probes (top left and right), whole-genome fragment input library (top middle) and hybrid selection-enriched output library (middle left and right). The captured DNA was treated with sodium bisulfite, amplified by PCR and sequenced using an Illumina GAIIx sequencer. ( b ) Examples of targeted bisulfite sequencing using solution hybrid selection. The tracks shown from the top to bottom are: sequences of the capture probes; the DNA methylation level at each CpG site derived from the bisulfite-sequencing reads; the sequencing depth at each CpG site; RefSeq genes; and annotated CGIs in the UCSC genome browser. Red and green colors indicate methylated and unmethylated-CpG sites, respectively. MCF7-1 and MCF7-2 are technical replicates. For each sample, two tracks (methylation level and read depth) are shown. ( c ). Distribution of normalized abundance for the captured targets shared among all samples. The x -axis is the normalized abundance of each captured target, which is calculated by dividing the counts of the target by the average counts of all targets. The y -axis is the fraction of probes with coverage equal to or greater than the normalized coverage.

Techniques Used: Methylation Sequencing, Selection, Amplification, Polymerase Chain Reaction, DNA Methylation Assay, Derivative Assay, Sequencing, Methylation

6) Product Images from "Sense Transgene-Induced Post-Transcriptional Gene Silencing in Tobacco Compromises the Splicing of Endogenous Counterpart Genes"

Article Title: Sense Transgene-Induced Post-Transcriptional Gene Silencing in Tobacco Compromises the Splicing of Endogenous Counterpart Genes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0087869

Expression levels of the endo-NtFAD3 gene. ( A ) The transcript levels of the endo-NtFAD3 gene in the total RNA fraction. Each endo-NtFAD3 transcript level was determined by qRT-PCR and normalized to the level of actin cDNA. The normalized value for the amount of endo-NtFAD3 mRNAs of the WT plants was considered to be 100%, and other normalized values in the S20, S44-hemi, and S44-homo plants were calculated as a percentage of that of the WT plants. e1-e2 and e8-e9 indicate the detection of endo-NtFAD3 transcripts that contain the proximal region (exon 1 to exon 2) and distal region (exon 8 to exon 9), respectively. e1-e7 indicates the detection of endo-NtFAD3 transcripts that contain a region from exon 1 to exon 7. The values are the mean ± SD (n = 3). ( B ) Pol II occupancy in the WT and S44 plants. ChIP-qPCR experiments were performed using an anti-Pol II antibody to detect the binding level at the indicated locus in the WT and the S44 plants. The mean of qPCR of the S44 sample is reported relative to the EF-1α gene control and is shown as a relative value of the corresponding WT level shown as 1.0. The graphical representation shows the fold change as the mean of the three different biological (and two different technical) replicates. The error bars represent SD. Different letters on the graph represent significantly different means ( P
Figure Legend Snippet: Expression levels of the endo-NtFAD3 gene. ( A ) The transcript levels of the endo-NtFAD3 gene in the total RNA fraction. Each endo-NtFAD3 transcript level was determined by qRT-PCR and normalized to the level of actin cDNA. The normalized value for the amount of endo-NtFAD3 mRNAs of the WT plants was considered to be 100%, and other normalized values in the S20, S44-hemi, and S44-homo plants were calculated as a percentage of that of the WT plants. e1-e2 and e8-e9 indicate the detection of endo-NtFAD3 transcripts that contain the proximal region (exon 1 to exon 2) and distal region (exon 8 to exon 9), respectively. e1-e7 indicates the detection of endo-NtFAD3 transcripts that contain a region from exon 1 to exon 7. The values are the mean ± SD (n = 3). ( B ) Pol II occupancy in the WT and S44 plants. ChIP-qPCR experiments were performed using an anti-Pol II antibody to detect the binding level at the indicated locus in the WT and the S44 plants. The mean of qPCR of the S44 sample is reported relative to the EF-1α gene control and is shown as a relative value of the corresponding WT level shown as 1.0. The graphical representation shows the fold change as the mean of the three different biological (and two different technical) replicates. The error bars represent SD. Different letters on the graph represent significantly different means ( P

Techniques Used: Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

Effects of the ectopic expression of the rgs-CaM gene on endo-NtFAD3 transcripts. ( A ) RT-PCR analysis of endo-NtFAD3 transcripts. The levels of endo-NtFAD3 transcripts were determined by RT-PCR analysis using the exon 2-specific and N3-AN primers. The equivalence of the amount of RNA used in RT-PCR is shown by the amplification of actin cDNA fragments. Two independent revertants (rv) and two independent non-revertants (nr) were identified among the descendants of a crossbred line (S44×CaM8) by measuring the leaf 18∶3 contents [23] . Three cDNA fragments found in the non-revertant lines were cloned and sequenced. ( B ) Structures of the endo-NtFAD3 transcripts generated in the non-revertant plants. The exons and introns are shown with open boxes and solid bars. The exon numbers are shown on the corresponding boxes.
Figure Legend Snippet: Effects of the ectopic expression of the rgs-CaM gene on endo-NtFAD3 transcripts. ( A ) RT-PCR analysis of endo-NtFAD3 transcripts. The levels of endo-NtFAD3 transcripts were determined by RT-PCR analysis using the exon 2-specific and N3-AN primers. The equivalence of the amount of RNA used in RT-PCR is shown by the amplification of actin cDNA fragments. Two independent revertants (rv) and two independent non-revertants (nr) were identified among the descendants of a crossbred line (S44×CaM8) by measuring the leaf 18∶3 contents [23] . Three cDNA fragments found in the non-revertant lines were cloned and sequenced. ( B ) Structures of the endo-NtFAD3 transcripts generated in the non-revertant plants. The exons and introns are shown with open boxes and solid bars. The exon numbers are shown on the corresponding boxes.

Techniques Used: Expressing, Chick Chorioallantoic Membrane Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Clone Assay, Generated

Detection of the 3′- and 5′-truncated endo-NtFAD3 transcripts. ( A ) RT-PCR analysis of the endo-NtFAD3 transcripts harboring the proximal region. S44-hemi and S44-homo denote the S44 plants hemizygous and homozygous for T-DNA, respectively. The amplified regions are illustrated. The endo-NtFAD3 mRNA is shown with open boxes. Each box with a number shows the corresponding exon. ( B ) 3′ RACE analysis. The total RNAs from the S20, S44-hemi, and S44-homo leaves were subjected to 3′ RACE. The RACE products from mature and truncated endo-NtFAD3 transcripts are indicated. The 3′ RACE products from truncated endo-NtFAD3 transcripts were cloned as follows: ten independent clones were sequenced and classified into 3 groups (a, b, and c) based on the positions of their polyadenylation sites. ( C ) RT-PCR analysis of the endo-NtFAD3 transcripts harboring the distal region. The amplified region is also illustrated, as in the case of Fig. 1A. ( D ) 5′ RACE analysis. The leaf total RNA was subjected to 5′ RACE. Five cDNA fragments (designated d to h) specific to the samples from the NtFAD3 transformants were cloned, and the structures of the 5′-truncated endo-NtFAD3 transcripts are shown. The open boxes and bars indicate exons and introns, respectively.
Figure Legend Snippet: Detection of the 3′- and 5′-truncated endo-NtFAD3 transcripts. ( A ) RT-PCR analysis of the endo-NtFAD3 transcripts harboring the proximal region. S44-hemi and S44-homo denote the S44 plants hemizygous and homozygous for T-DNA, respectively. The amplified regions are illustrated. The endo-NtFAD3 mRNA is shown with open boxes. Each box with a number shows the corresponding exon. ( B ) 3′ RACE analysis. The total RNAs from the S20, S44-hemi, and S44-homo leaves were subjected to 3′ RACE. The RACE products from mature and truncated endo-NtFAD3 transcripts are indicated. The 3′ RACE products from truncated endo-NtFAD3 transcripts were cloned as follows: ten independent clones were sequenced and classified into 3 groups (a, b, and c) based on the positions of their polyadenylation sites. ( C ) RT-PCR analysis of the endo-NtFAD3 transcripts harboring the distal region. The amplified region is also illustrated, as in the case of Fig. 1A. ( D ) 5′ RACE analysis. The leaf total RNA was subjected to 5′ RACE. Five cDNA fragments (designated d to h) specific to the samples from the NtFAD3 transformants were cloned, and the structures of the 5′-truncated endo-NtFAD3 transcripts are shown. The open boxes and bars indicate exons and introns, respectively.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Clone Assay

7) Product Images from "Transcriptomic and GC-MS Metabolomic Analyses Reveal the Sink Strength Changes during Petunia Anther Development"

Article Title: Transcriptomic and GC-MS Metabolomic Analyses Reveal the Sink Strength Changes during Petunia Anther Development

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19040955

The qRT-PCR validation of DEGs. The relative expression levels of 18 transcription factors ( A ), 14 starch and sucrose metabolism pathways ( B ) and 6 photosynthesis related genes ( C ). The left Y axis represents the relative transcript amount obtained by qRT-PCR. The right Y axis represents the fragments per kb per million fragments (FPKM) value of each gene using RNA-Seq analysis. Error bars indicate the standard errors. ( D ) Correlation analysis of the gene expression ratios between qRT-PCR and RNA-seq.
Figure Legend Snippet: The qRT-PCR validation of DEGs. The relative expression levels of 18 transcription factors ( A ), 14 starch and sucrose metabolism pathways ( B ) and 6 photosynthesis related genes ( C ). The left Y axis represents the relative transcript amount obtained by qRT-PCR. The right Y axis represents the fragments per kb per million fragments (FPKM) value of each gene using RNA-Seq analysis. Error bars indicate the standard errors. ( D ) Correlation analysis of the gene expression ratios between qRT-PCR and RNA-seq.

Techniques Used: Quantitative RT-PCR, Expressing, RNA Sequencing Assay

8) Product Images from "Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo"

Article Title: Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo

Journal: Genome Biology

doi: 10.1186/gb-2014-15-5-r75

Barcode plasmid library analysis. Results from four separate PCR amplification and sequencing runs of the plasmid barcode library (A to D). (a) The number of barcodes found in each replicate after analysis and trimming. 'Mean' is the average number of barcodes for the four replicates; 'Total' is the number of unique barcodes found within the four samples combined. (b) Venn diagram demonstrating the amount of overlap of barcodes among the four replicates. Darker shading indicates larger numbers of barcodes. (c) Barcodes were counted and grouped in Log 2 bins based on percentage (frequency) within the population, from least to greatest. The percentage of the barcodes in each bin is shown. (d) The predicted (Expected) and experimentally determined median and mean barcode frequencies are shown as percentages, as well as the standard deviation from the mean. (e) The percentage of barcodes, ranked from most to least frequent plotted by what percentage of the total sequences they made up. Dashed line represents perfectly equal representation of barcodes. (f) The percentage of sequences made up by the top indicated percentages of the barcodes for each sample.
Figure Legend Snippet: Barcode plasmid library analysis. Results from four separate PCR amplification and sequencing runs of the plasmid barcode library (A to D). (a) The number of barcodes found in each replicate after analysis and trimming. 'Mean' is the average number of barcodes for the four replicates; 'Total' is the number of unique barcodes found within the four samples combined. (b) Venn diagram demonstrating the amount of overlap of barcodes among the four replicates. Darker shading indicates larger numbers of barcodes. (c) Barcodes were counted and grouped in Log 2 bins based on percentage (frequency) within the population, from least to greatest. The percentage of the barcodes in each bin is shown. (d) The predicted (Expected) and experimentally determined median and mean barcode frequencies are shown as percentages, as well as the standard deviation from the mean. (e) The percentage of barcodes, ranked from most to least frequent plotted by what percentage of the total sequences they made up. Dashed line represents perfectly equal representation of barcodes. (f) The percentage of sequences made up by the top indicated percentages of the barcodes for each sample.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Sequencing, Standard Deviation

9) Product Images from "Transcription elongation factors represent in vivo cancer dependencies in glioblastoma"

Article Title: Transcription elongation factors represent in vivo cancer dependencies in glioblastoma

Journal: Nature

doi: 10.1038/nature23000

Validation of JMJD6 and other hits in multiple PDX models of glioblastoma a , JMJD6 mRNA expression by qRT-PCR after inducible shRNA knockdown of JMJD6. b , In vitro proliferation and c , in vivo survival compared to uninduced and induced non-targeting controls. Values are mean +/− s.d. of 3 technical replicates. d, Endpoint tumours harvested from the induced arm of ( c ) stained to show human tumour cells (human nuclear antigen) that harbour a JMJD6 shRNA (Venus+) or harbour and express a JMJD6 shRNA (Venus+dsRED+). The vast majority of tumour cells at endpoint had silenced the shRNA (Venus+dsRED−). Scale bar: 200 μM. e , CRISPR mediated knockout of JMJD6 in a bulk population of GBMcw1919 cells in vitro. f–g , Parallel in vitro proliferation assay ( f ) and in vivo survival assay ( g ) of cells from ( e ). h–l , Constitutive shRNA knockdown of DOT1L and DPY30 in vitro. m–v , Parallel in vitro proliferation assays ( m–q ) and in vivo survival assays ( r–v ) of cells from ( h–l ), respectively. Error bars of bar graphs +/− s.d. of at least triplicates.
Figure Legend Snippet: Validation of JMJD6 and other hits in multiple PDX models of glioblastoma a , JMJD6 mRNA expression by qRT-PCR after inducible shRNA knockdown of JMJD6. b , In vitro proliferation and c , in vivo survival compared to uninduced and induced non-targeting controls. Values are mean +/− s.d. of 3 technical replicates. d, Endpoint tumours harvested from the induced arm of ( c ) stained to show human tumour cells (human nuclear antigen) that harbour a JMJD6 shRNA (Venus+) or harbour and express a JMJD6 shRNA (Venus+dsRED+). The vast majority of tumour cells at endpoint had silenced the shRNA (Venus+dsRED−). Scale bar: 200 μM. e , CRISPR mediated knockout of JMJD6 in a bulk population of GBMcw1919 cells in vitro. f–g , Parallel in vitro proliferation assay ( f ) and in vivo survival assay ( g ) of cells from ( e ). h–l , Constitutive shRNA knockdown of DOT1L and DPY30 in vitro. m–v , Parallel in vitro proliferation assays ( m–q ) and in vivo survival assays ( r–v ) of cells from ( h–l ), respectively. Error bars of bar graphs +/− s.d. of at least triplicates.

Techniques Used: Expressing, Quantitative RT-PCR, shRNA, In Vitro, In Vivo, Staining, CRISPR, Knock-Out, Proliferation Assay, Clonogenic Cell Survival Assay

10) Product Images from "Novel Discovery of LINE-1 in a Korean Individual by a Target Enrichment Method"

Article Title: Novel Discovery of LINE-1 in a Korean Individual by a Target Enrichment Method

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0351

The workflow of L1Hs-targeted enrichment library preparation (A) Double-strand genomic DNA (blue) is extracted from a Korean individual genome (KPGP9). Red boxes indicate the regions where the probe binds to the 3′ UTR of L1Hs elements. (B) Genomic DNA is fragmented by aquatic ultrasonic wave of the Covaris S2 system. Sheared DNAs have an average size of 550 bp, which is suitable for HiSeq sequencing. (C) The Illumina’s adaptor (green) is ligated at both ends of the fragmented DNAs. (D) The adaptor-ligated DNAs is hybridized with the L1Hs-targeted probe (red and orange). Only the presence of the L1Hs 3′ UTR allows the sequence-specific binding of the probe. (E) Targeted DNA fragments are selectively elongated from the probe-binding strands. (F) Because the probe sequence attached to the L1Hs 3′ UTR and the Illumina’s adaptor sequences at both ends are known, targeted DNAs are enriched by PCR with the primer set. After library construction, the final product is confirmed using the Agilent Bioanalyzer High Sensitivity chip assay.
Figure Legend Snippet: The workflow of L1Hs-targeted enrichment library preparation (A) Double-strand genomic DNA (blue) is extracted from a Korean individual genome (KPGP9). Red boxes indicate the regions where the probe binds to the 3′ UTR of L1Hs elements. (B) Genomic DNA is fragmented by aquatic ultrasonic wave of the Covaris S2 system. Sheared DNAs have an average size of 550 bp, which is suitable for HiSeq sequencing. (C) The Illumina’s adaptor (green) is ligated at both ends of the fragmented DNAs. (D) The adaptor-ligated DNAs is hybridized with the L1Hs-targeted probe (red and orange). Only the presence of the L1Hs 3′ UTR allows the sequence-specific binding of the probe. (E) Targeted DNA fragments are selectively elongated from the probe-binding strands. (F) Because the probe sequence attached to the L1Hs 3′ UTR and the Illumina’s adaptor sequences at both ends are known, targeted DNAs are enriched by PCR with the primer set. After library construction, the final product is confirmed using the Agilent Bioanalyzer High Sensitivity chip assay.

Techniques Used: Sequencing, Binding Assay, Polymerase Chain Reaction, Chromatin Immunoprecipitation

11) Product Images from "Megabase length hypermutation accompanies human structural variation at 17p11.2"

Article Title: Megabase length hypermutation accompanies human structural variation at 17p11.2

Journal: Cell

doi: 10.1016/j.cell.2019.01.045

SNVs and indels accompany SV formation A) BAB2543 carries two duplications in an inverted orientation separated with a copy-neutral segment (DUP-NML-DUP/INV). Breakpoint junction (jct) 1 maps to inverted SMSREP LCRs, and evaded sequencing attempts. Breakpoint jct 2 was mediated by inverted Alu repeats and forms an Alu-Alu chimera; junction sequence is characterized by 29 bp of microhomology. Eight de novo mutations have also been characterized within 17p11.2; Sanger sequencing electropherogram confirming each SNV is shown along with the location, genomic context and type. B) BAB1931 carries three deletions interspersed with copy-neutral segments (DEL-NML-DEL-NML-DEL). SV breakpoints display one, two and zero bp of microhomology at the junctions, and jct3 was previously uncharacterized. The four de novo SNVs and indels present in the proband and Sanger sequencing electropherogram confirmation are depicted below. The SNV at 20409881 was not independently confirmed by using a PCR/Sanger sequencing strategy due to its presence within an LCR; however, it was observed in both Illumina and PacBio sequencing data and shown to be de novo in the trio Illumina sequencing data. C) Plot shows the relative contribution of each SNV transition and transversion observed de novo in the non-recurrent individuals. Overall abundance of C > G mutations can be readily observed. D) Enrichment of de novo SNVs in proximity to SV breakpoints was observed in the genomes of 9 out of 13 subjects with non-recurrent SV. This enrichment was not observed for de novo SNVs (N=4) detected in the subjects carrying recurrent SVs. The normalized statistics (Z-value) for each simulation and observation (red dot) is displayed with the box plots. E) .
Figure Legend Snippet: SNVs and indels accompany SV formation A) BAB2543 carries two duplications in an inverted orientation separated with a copy-neutral segment (DUP-NML-DUP/INV). Breakpoint junction (jct) 1 maps to inverted SMSREP LCRs, and evaded sequencing attempts. Breakpoint jct 2 was mediated by inverted Alu repeats and forms an Alu-Alu chimera; junction sequence is characterized by 29 bp of microhomology. Eight de novo mutations have also been characterized within 17p11.2; Sanger sequencing electropherogram confirming each SNV is shown along with the location, genomic context and type. B) BAB1931 carries three deletions interspersed with copy-neutral segments (DEL-NML-DEL-NML-DEL). SV breakpoints display one, two and zero bp of microhomology at the junctions, and jct3 was previously uncharacterized. The four de novo SNVs and indels present in the proband and Sanger sequencing electropherogram confirmation are depicted below. The SNV at 20409881 was not independently confirmed by using a PCR/Sanger sequencing strategy due to its presence within an LCR; however, it was observed in both Illumina and PacBio sequencing data and shown to be de novo in the trio Illumina sequencing data. C) Plot shows the relative contribution of each SNV transition and transversion observed de novo in the non-recurrent individuals. Overall abundance of C > G mutations can be readily observed. D) Enrichment of de novo SNVs in proximity to SV breakpoints was observed in the genomes of 9 out of 13 subjects with non-recurrent SV. This enrichment was not observed for de novo SNVs (N=4) detected in the subjects carrying recurrent SVs. The normalized statistics (Z-value) for each simulation and observation (red dot) is displayed with the box plots. E) .

Techniques Used: Sequencing, Polymerase Chain Reaction

12) Product Images from "The histone chaperone CAF-1 safeguards somatic cell identity"

Article Title: The histone chaperone CAF-1 safeguards somatic cell identity

Journal: Nature

doi: 10.1038/nature15749

Confirmation of CAF-1 reprogramming phenotype with alternative transgenic and non-transgenic vector systems (a) Alkaline phosphatase (AP)-positive, transgene-independent iPSC colonies at day 14 following transduction of R26-M2rtTA MEFs with tetO-STEMCCA lentiviral OKSM expression vector and either Chaf1a.164 or Ren.713 shRNA vectors and treatment with high (2 μg/ml) or low (0.2 μg/ml) doses of dox for 10 days. (b) Quantification of data shown in (a). Experiment was performed at 3 different plating densities (n=1 experiment per density), representative data are shown. (c) Comparison of reprogramming efficiencies between Col1a1::tetOP-OKSM; R26-M2rtTA reprogrammable MEFs and wild type MEFs infected directly with OKSM-expressing lentiviral vectors containing either a strong Ef1a full-length promoter (Ef1a-OKSM long) or a weaker truncated promoter (Ef1a-OKSM short). TRE3G-OKSM is a lentiviral vector with a strong promoter, whose activity is downregulated over time upon infection of CAGS-rtTA3 transgenic MEFs (see below). Error bars show standard deviation from biological triplicates. (d) Quantitative RT-PCR data showing variability in OKSM expression levels over time using different vector systems. Cells were analyzed after 3 and 6 days of infection (lentiviral vectors) or dox exposure (reprogrammable MEFs). Error bars show standard deviation from biological triplicates. OGR MEF, transgenic MEFs carrying O ct4- G FP and CAGS- r tTA3 alleles. (e) Quantification of Oct4 protein levels by intracellular flow cytometry (top) and cellular granularity/complexity by side scatter (SSC) analysis of indicated samples (bottom). Error bars show standard deviation from biological triplicates.
Figure Legend Snippet: Confirmation of CAF-1 reprogramming phenotype with alternative transgenic and non-transgenic vector systems (a) Alkaline phosphatase (AP)-positive, transgene-independent iPSC colonies at day 14 following transduction of R26-M2rtTA MEFs with tetO-STEMCCA lentiviral OKSM expression vector and either Chaf1a.164 or Ren.713 shRNA vectors and treatment with high (2 μg/ml) or low (0.2 μg/ml) doses of dox for 10 days. (b) Quantification of data shown in (a). Experiment was performed at 3 different plating densities (n=1 experiment per density), representative data are shown. (c) Comparison of reprogramming efficiencies between Col1a1::tetOP-OKSM; R26-M2rtTA reprogrammable MEFs and wild type MEFs infected directly with OKSM-expressing lentiviral vectors containing either a strong Ef1a full-length promoter (Ef1a-OKSM long) or a weaker truncated promoter (Ef1a-OKSM short). TRE3G-OKSM is a lentiviral vector with a strong promoter, whose activity is downregulated over time upon infection of CAGS-rtTA3 transgenic MEFs (see below). Error bars show standard deviation from biological triplicates. (d) Quantitative RT-PCR data showing variability in OKSM expression levels over time using different vector systems. Cells were analyzed after 3 and 6 days of infection (lentiviral vectors) or dox exposure (reprogrammable MEFs). Error bars show standard deviation from biological triplicates. OGR MEF, transgenic MEFs carrying O ct4- G FP and CAGS- r tTA3 alleles. (e) Quantification of Oct4 protein levels by intracellular flow cytometry (top) and cellular granularity/complexity by side scatter (SSC) analysis of indicated samples (bottom). Error bars show standard deviation from biological triplicates.

Techniques Used: Transgenic Assay, Plasmid Preparation, Transduction, Expressing, shRNA, Infection, Activity Assay, Standard Deviation, Quantitative RT-PCR, Flow Cytometry, Cytometry

Effect of CAF-1 suppression on HSPC reprogramming and transdifferentiation (a) Gating strategy for determining Pecam + fraction (shaded area) in panel (b); data identical to Fig. 4c . (b) Quantification of the fraction of Pecam + cells at day 4 and day 6 of reprogramming. Data obtained from one experiment using 2 different Chaf1 shRNAs. (c) Transgene dependence assay during the reprogramming of hematopoietic stem and progenitor cells (HSPCs) into iPSCs in the presence of Chaf1a or Renilla shRNAs. Dox pulses were given for 3 or 6 days and alkaline phosphatase (AP)-positive colonies were scored at day 10. (d) Quantitative RT-PCR analysis of Chaf1a expression to confirm knockdown after 3 days of dox induction, i.e. coexpression of shRNAmiR and Ascl1 (n=4 independent infections of the same Col1a1::tetOP-Chaf1a.164 shRNA MEF line; mean value +/− standard deviation). (e) Gating strategy for determining Cd14 + and Mac1 + fractions (shaded area) shown in (f); data identical to Fig. 4g . Positive gates were based on untreated (0 hour) control cells. (f) Quantification of the fraction of Cd14 + and Mac1 + cells at 0, 24 and 48 hours of transdifferentiation using indicated CAF-1 shRNA or empty control vector (n=2 independent infections; rep, replicate). (g) Quantitative RT-PCR analysis of Chaf1a and Chaf1b expression to confirm knockdown in transduced pre-B cell line prior to induction of transdifferentiation (kd/ctrl, knockdown/empty vector control; n=1 experiment, representative of 2 independent infections).
Figure Legend Snippet: Effect of CAF-1 suppression on HSPC reprogramming and transdifferentiation (a) Gating strategy for determining Pecam + fraction (shaded area) in panel (b); data identical to Fig. 4c . (b) Quantification of the fraction of Pecam + cells at day 4 and day 6 of reprogramming. Data obtained from one experiment using 2 different Chaf1 shRNAs. (c) Transgene dependence assay during the reprogramming of hematopoietic stem and progenitor cells (HSPCs) into iPSCs in the presence of Chaf1a or Renilla shRNAs. Dox pulses were given for 3 or 6 days and alkaline phosphatase (AP)-positive colonies were scored at day 10. (d) Quantitative RT-PCR analysis of Chaf1a expression to confirm knockdown after 3 days of dox induction, i.e. coexpression of shRNAmiR and Ascl1 (n=4 independent infections of the same Col1a1::tetOP-Chaf1a.164 shRNA MEF line; mean value +/− standard deviation). (e) Gating strategy for determining Cd14 + and Mac1 + fractions (shaded area) shown in (f); data identical to Fig. 4g . Positive gates were based on untreated (0 hour) control cells. (f) Quantification of the fraction of Cd14 + and Mac1 + cells at 0, 24 and 48 hours of transdifferentiation using indicated CAF-1 shRNA or empty control vector (n=2 independent infections; rep, replicate). (g) Quantitative RT-PCR analysis of Chaf1a and Chaf1b expression to confirm knockdown in transduced pre-B cell line prior to induction of transdifferentiation (kd/ctrl, knockdown/empty vector control; n=1 experiment, representative of 2 independent infections).

Techniques Used: Quantitative RT-PCR, Expressing, shRNA, Standard Deviation, Plasmid Preparation

Effect of CAF-1 suppression on OKSM levels and cellular growth, and shRNA rescue experiment (a) Quantitative RT-PCR for transgenic OKSM expression using reprogrammable MEFs transduced with indicated shRNA vectors. Error bars show standard deviation from biological triplicates. (b) RNA-seq analysis of OKSM transgene expression in reprogrammable MEFs transduced with Renilla and Chaf1a shRNAs and exposed to dox for 0, 3 or 6 days. Error bars indicate standard deviation from biological triplicates. (c) Western blot analysis for Sox2 and Tbp (loading control) in reprogrammable MEFs transduced with shRNA vectors targeting Renilla (Ren.713) or different CAF-1 components and exposed to dox for 3 days (see Supplementary Figure 1 for full scans). The same membrane was probed with anti-CAF-1 p150 and anti-CAF-1 p60 antibody to confirm knockdown (data not shown). (d) Rescue experiment to demonstrate specificity of Chaf1b.367 shRNA vector. Reprogrammable MEFs carrying Oct4-tomato knock-in reporter were infected with lentiviral vectors expressing either EGFP or human CAF-1 p60 (CHAF1B) before transducing cells with Renilla or Cha1fb.367 shRNAs and applying dox for 6 days. Colonies were counted at day 11. Note that CAF-1 p60 overexpression attenuates enhanced reprogramming elicited by Chaf1b suppression. (e,f) Competitive proliferation assay between shRNA vector-infected and non-infected reprogrammable cells using indicated shRNAs in the presence or absence of dox (OKSM expression). Note that CAF-1 suppression does not substantially affect the proliferation potential of reprogrammable MEFs after 1-3 days of dox (OKSM) induction while it impairs the long-term growth potential of uninduced MEFs. Data were normalized to cell counts in “no OKSM” condition for (e) and “day 2” time point for (f). Error bars show standard deviation from biological triplicates.
Figure Legend Snippet: Effect of CAF-1 suppression on OKSM levels and cellular growth, and shRNA rescue experiment (a) Quantitative RT-PCR for transgenic OKSM expression using reprogrammable MEFs transduced with indicated shRNA vectors. Error bars show standard deviation from biological triplicates. (b) RNA-seq analysis of OKSM transgene expression in reprogrammable MEFs transduced with Renilla and Chaf1a shRNAs and exposed to dox for 0, 3 or 6 days. Error bars indicate standard deviation from biological triplicates. (c) Western blot analysis for Sox2 and Tbp (loading control) in reprogrammable MEFs transduced with shRNA vectors targeting Renilla (Ren.713) or different CAF-1 components and exposed to dox for 3 days (see Supplementary Figure 1 for full scans). The same membrane was probed with anti-CAF-1 p150 and anti-CAF-1 p60 antibody to confirm knockdown (data not shown). (d) Rescue experiment to demonstrate specificity of Chaf1b.367 shRNA vector. Reprogrammable MEFs carrying Oct4-tomato knock-in reporter were infected with lentiviral vectors expressing either EGFP or human CAF-1 p60 (CHAF1B) before transducing cells with Renilla or Cha1fb.367 shRNAs and applying dox for 6 days. Colonies were counted at day 11. Note that CAF-1 p60 overexpression attenuates enhanced reprogramming elicited by Chaf1b suppression. (e,f) Competitive proliferation assay between shRNA vector-infected and non-infected reprogrammable cells using indicated shRNAs in the presence or absence of dox (OKSM expression). Note that CAF-1 suppression does not substantially affect the proliferation potential of reprogrammable MEFs after 1-3 days of dox (OKSM) induction while it impairs the long-term growth potential of uninduced MEFs. Data were normalized to cell counts in “no OKSM” condition for (e) and “day 2” time point for (f). Error bars show standard deviation from biological triplicates.

Techniques Used: shRNA, Quantitative RT-PCR, Transgenic Assay, Expressing, Transduction, Standard Deviation, RNA Sequencing Assay, Western Blot, Plasmid Preparation, Knock-In, Infection, Over Expression, Proliferation Assay

Validation of hits from chromatin-focused shRNA screens (a) Quantitative RT-PCR analysis to confirm suppression of Chaf1a and Chaf1b expression with miR-30-based vectors from arrayed screen. Sh Chaf1a pool, sh Chaf1b pool and sh CAF-1 pool denote pools of shRNAs targeting either Chaf1a, Chaf1b or both. (b) Western blot analysis to confirm knockdown of CAF-1 components using the top-scoring miR-30-based shRNAs from arrayed screen (see Supplementary Figure 1 for full scans). (c) Quantification of data shown in Fig. 1f . (d) Quantitative RT-PCR analysis confirming knockdown with top-scoring miR-E-based shRNAmiRs targeting Chaf1a, Chaf1b or Ube2i from multiplexed screen. Error bars show standard deviation from biological triplicates. RNA and protein were extracted from reprogrammable MEFs 72 hours after dox induction in panels a-d. (e) Suppression of CAF-1 components, Ube2i and Setdb2 enhances reprogramming in the presence or absence of ascorbic acid (AA) as well as in serum replacement media containing LIF (SR-LIF). Oct4-GFP + cells were scored by flow cytometry on day 11 after 7 days of OKSM induction and 4 days of transgene-independent growth. Error bars show standard deviation from biological triplicates. (f) Number of dox-independent, alkaline phosphatase (AP)-positive colonies emerging 2 weeks after plating 10,000 reprogrammable MEFs carrying shRNA vectors against indicated targets and cultured in serum replacement media containing 2i (SR-2i), n=1 experiment. (g) Effect of suppressing SUMO E2 ligase Ube2i, E1 ligases Sae1 and Uba2 on iPSC formation. Shown is fraction of Oct4-GFP + cells at day 11 (7 days of OKSM induction, 4 days of transgene-independent growth). Error bars depict standard deviation from biological triplicates.
Figure Legend Snippet: Validation of hits from chromatin-focused shRNA screens (a) Quantitative RT-PCR analysis to confirm suppression of Chaf1a and Chaf1b expression with miR-30-based vectors from arrayed screen. Sh Chaf1a pool, sh Chaf1b pool and sh CAF-1 pool denote pools of shRNAs targeting either Chaf1a, Chaf1b or both. (b) Western blot analysis to confirm knockdown of CAF-1 components using the top-scoring miR-30-based shRNAs from arrayed screen (see Supplementary Figure 1 for full scans). (c) Quantification of data shown in Fig. 1f . (d) Quantitative RT-PCR analysis confirming knockdown with top-scoring miR-E-based shRNAmiRs targeting Chaf1a, Chaf1b or Ube2i from multiplexed screen. Error bars show standard deviation from biological triplicates. RNA and protein were extracted from reprogrammable MEFs 72 hours after dox induction in panels a-d. (e) Suppression of CAF-1 components, Ube2i and Setdb2 enhances reprogramming in the presence or absence of ascorbic acid (AA) as well as in serum replacement media containing LIF (SR-LIF). Oct4-GFP + cells were scored by flow cytometry on day 11 after 7 days of OKSM induction and 4 days of transgene-independent growth. Error bars show standard deviation from biological triplicates. (f) Number of dox-independent, alkaline phosphatase (AP)-positive colonies emerging 2 weeks after plating 10,000 reprogrammable MEFs carrying shRNA vectors against indicated targets and cultured in serum replacement media containing 2i (SR-2i), n=1 experiment. (g) Effect of suppressing SUMO E2 ligase Ube2i, E1 ligases Sae1 and Uba2 on iPSC formation. Shown is fraction of Oct4-GFP + cells at day 11 (7 days of OKSM induction, 4 days of transgene-independent growth). Error bars depict standard deviation from biological triplicates.

Techniques Used: shRNA, Quantitative RT-PCR, Expressing, Western Blot, Standard Deviation, Flow Cytometry, Cytometry, Cell Culture

13) Product Images from "Clinical molecular testing for ASXL1 c.1934dupG p.Gly646fs mutation in hematologic neoplasms in the NGS era"

Article Title: Clinical molecular testing for ASXL1 c.1934dupG p.Gly646fs mutation in hematologic neoplasms in the NGS era

Journal: PLoS ONE

doi: 10.1371/journal.pone.0204218

A . VAF scatterplot plotting all the clinical samples studied by NGS with direct PCR amplification (PA) based library preparation chemistry found to harbor ASXL1 c.1934dupG (n = 2870). Majority of the cases (2636, 92%) have a variant frequency below 10%. Y axis: VAF. B . 234 samples (8%) were identified with ASXL1 c.1934dupG with VAF≥10%. The maximum VAF observed in the series was 51.4. X axis: VAF, Y axis: number of samples. C . VAF scatterplot showing all the clinical samples studied by HCA-based NGS library preparation chemistry found to harbor ASXL1 c.1934dupG (n = 250).Y axis: VAF D . Left panel: VAF scatterplot showing all the clinical samples studied by NGS with amplicon based library preparation chemistry found to harbor ASXL1 c.1934del p.G645fs (n = 2864). As depicted, most of the cases (99%) have a variant frequency below 10%. The maximum VAF observed in the series was 40.36. Y axis: VAF. Right panel: VAF scatterplot showing all the clinical samples studied by HCA-based NGS found to harbor ASXL1 c.1934del p.G645fs (n = 74). Y axis: VAF. E . Three electropherograms of cases with ASXL1 c.1934dupG quantified as 3.59, 7.78 and 30.31 respectively by NGS after amplicon based library preparation. As shown, no minor clonal sequence is found in the case with a VAF
Figure Legend Snippet: A . VAF scatterplot plotting all the clinical samples studied by NGS with direct PCR amplification (PA) based library preparation chemistry found to harbor ASXL1 c.1934dupG (n = 2870). Majority of the cases (2636, 92%) have a variant frequency below 10%. Y axis: VAF. B . 234 samples (8%) were identified with ASXL1 c.1934dupG with VAF≥10%. The maximum VAF observed in the series was 51.4. X axis: VAF, Y axis: number of samples. C . VAF scatterplot showing all the clinical samples studied by HCA-based NGS library preparation chemistry found to harbor ASXL1 c.1934dupG (n = 250).Y axis: VAF D . Left panel: VAF scatterplot showing all the clinical samples studied by NGS with amplicon based library preparation chemistry found to harbor ASXL1 c.1934del p.G645fs (n = 2864). As depicted, most of the cases (99%) have a variant frequency below 10%. The maximum VAF observed in the series was 40.36. Y axis: VAF. Right panel: VAF scatterplot showing all the clinical samples studied by HCA-based NGS found to harbor ASXL1 c.1934del p.G645fs (n = 74). Y axis: VAF. E . Three electropherograms of cases with ASXL1 c.1934dupG quantified as 3.59, 7.78 and 30.31 respectively by NGS after amplicon based library preparation. As shown, no minor clonal sequence is found in the case with a VAF

Techniques Used: Next-Generation Sequencing, Polymerase Chain Reaction, Amplification, Variant Assay, High Content Screening, Sequencing

14) Product Images from "Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation"

Article Title: Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx286

Schematic overview of Poly(A)ClickSeq (PAC-seq). ( A ) RT-PCR is launched from a non-anchored Poly(T) primer containing a portion of the Illumina p7 adaptor. RT-PCR is performed in the presence of AzATP, AzGTP and AzCTP, but not AzTTP, thus only allowing chain termination to occur upstream of the poly(A) tail in the 3΄UTR. ( B ) 3΄-Azido-blocked cDNA fragments are ‘click-ligated’ to 5΄-hexynyl–functionalised DNA oligos containing the p5 Illumina adaptor. This yields triazole-linked ssDNA which can be PCR-amplified using primers to the p5 and p7 Illumina adaptors. ( C ) The cDNA library is analysed by gel electrophoresis and should consist of a smear of DNA products centered ∼200–300 bp. Appropriate cDNA fragment sizes are cut out of the gel and purified to yield a final library. ( D ) The final library consists of DNA fragments containing the Illumina p5 adaptor, a portion of the 3΄UTR, a stretch of As derived from both the RNA template and the poly(T) primer, and finally the p7 Illumina Indexing primer.
Figure Legend Snippet: Schematic overview of Poly(A)ClickSeq (PAC-seq). ( A ) RT-PCR is launched from a non-anchored Poly(T) primer containing a portion of the Illumina p7 adaptor. RT-PCR is performed in the presence of AzATP, AzGTP and AzCTP, but not AzTTP, thus only allowing chain termination to occur upstream of the poly(A) tail in the 3΄UTR. ( B ) 3΄-Azido-blocked cDNA fragments are ‘click-ligated’ to 5΄-hexynyl–functionalised DNA oligos containing the p5 Illumina adaptor. This yields triazole-linked ssDNA which can be PCR-amplified using primers to the p5 and p7 Illumina adaptors. ( C ) The cDNA library is analysed by gel electrophoresis and should consist of a smear of DNA products centered ∼200–300 bp. Appropriate cDNA fragment sizes are cut out of the gel and purified to yield a final library. ( D ) The final library consists of DNA fragments containing the Illumina p5 adaptor, a portion of the 3΄UTR, a stretch of As derived from both the RNA template and the poly(T) primer, and finally the p7 Illumina Indexing primer.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, cDNA Library Assay, Nucleic Acid Electrophoresis, Purification, Derivative Assay

15) Product Images from "Evaluation of relative quantification of alternatively spliced transcripts using droplet digital PCR"

Article Title: Evaluation of relative quantification of alternatively spliced transcripts using droplet digital PCR

Journal: Biomolecular Detection and Quantification

doi: 10.1016/j.bdq.2017.09.001

Linearity and dynamic range was tested on for all assays with both quantitative PCR methods, using dilution series containing transcript-specific plasmids ((a) and (b)) or cDNA from EBV samples (non-carriers ((c) and (d)) and carriers ((e) and (f)). Measured relative template quantity (RQ) is depicted on the y-axis in function of theoretical template concentration on the x-axis. Almost all assay-sample combinations show excellent linearity with both techniques (R 2 > 0.98). Linearity was only worse for BRCA1-Δex5 in dilutions from EBV controls with ddPCR (R 2 = 0.81). An important difference is that the dynamic range of qPCR across the dilution series was larger for several sample-target combinations, especially when quantifying transcript-specific plasmids. From these data we also estimated the LOB, LOD and LOQ values. For both methods LOB was equal to 0 (none of the no template controls yielded any amplification) and the LOQ and LOD for both methods reaches the theoretical minimum of 1 copy per reaction in transcript-specific plasmid dilution series. When using EBV derived samples the LOQ for both qPCR and ddPCR was found to range between 100 pg and 6.25 ng of cDNA depending on the abundance of a certain transcript in each sample.
Figure Legend Snippet: Linearity and dynamic range was tested on for all assays with both quantitative PCR methods, using dilution series containing transcript-specific plasmids ((a) and (b)) or cDNA from EBV samples (non-carriers ((c) and (d)) and carriers ((e) and (f)). Measured relative template quantity (RQ) is depicted on the y-axis in function of theoretical template concentration on the x-axis. Almost all assay-sample combinations show excellent linearity with both techniques (R 2 > 0.98). Linearity was only worse for BRCA1-Δex5 in dilutions from EBV controls with ddPCR (R 2 = 0.81). An important difference is that the dynamic range of qPCR across the dilution series was larger for several sample-target combinations, especially when quantifying transcript-specific plasmids. From these data we also estimated the LOB, LOD and LOQ values. For both methods LOB was equal to 0 (none of the no template controls yielded any amplification) and the LOQ and LOD for both methods reaches the theoretical minimum of 1 copy per reaction in transcript-specific plasmid dilution series. When using EBV derived samples the LOQ for both qPCR and ddPCR was found to range between 100 pg and 6.25 ng of cDNA depending on the abundance of a certain transcript in each sample.

Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Amplification, Plasmid Preparation, Derivative Assay

Boxplot of coefficients of variation for all cDNA-assay combinations over all time points of both quantitative PCR techniques. We observed an inverse correlation between the CV and the relative abundance of each isoform; CVs are consistently higher for isoforms with lower expression. CVs for each transcript range within the same order of magnitude and all experiments show very small standard errors, leading us to conclude that precision, reproducibility and repeatability are not an issue with either quantitative PCR technique.
Figure Legend Snippet: Boxplot of coefficients of variation for all cDNA-assay combinations over all time points of both quantitative PCR techniques. We observed an inverse correlation between the CV and the relative abundance of each isoform; CVs are consistently higher for isoforms with lower expression. CVs for each transcript range within the same order of magnitude and all experiments show very small standard errors, leading us to conclude that precision, reproducibility and repeatability are not an issue with either quantitative PCR technique.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

16) Product Images from "Transcriptome profiling of Elymus sibiricus, an important forage grass in Qinghai-Tibet plateau, reveals novel insights into candidate genes that potentially connected to seed shattering"

Article Title: Transcriptome profiling of Elymus sibiricus, an important forage grass in Qinghai-Tibet plateau, reveals novel insights into candidate genes that potentially connected to seed shattering

Journal: BMC Plant Biology

doi: 10.1186/s12870-017-1026-2

qRT-PCR validations of RNA-seq data. Expression profiles of the selected genes as determined by RNA-seq and qRT-PCR. Data were collected from high seed shattering genotype XH09 and low seed shattering genotype ZhN03 at 7, 21 and 28 days after heading. The left-hand y-axis indicates FPKM value. The right-hand y-axis indicates relative expression level. Bars indicate the mean values ± standard deviation
Figure Legend Snippet: qRT-PCR validations of RNA-seq data. Expression profiles of the selected genes as determined by RNA-seq and qRT-PCR. Data were collected from high seed shattering genotype XH09 and low seed shattering genotype ZhN03 at 7, 21 and 28 days after heading. The left-hand y-axis indicates FPKM value. The right-hand y-axis indicates relative expression level. Bars indicate the mean values ± standard deviation

Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay, Expressing, Standard Deviation

17) Product Images from "Deep Panning: Steps towards Probing the IgOme"

Article Title: Deep Panning: Steps towards Probing the IgOme

Journal: PLoS ONE

doi: 10.1371/journal.pone.0041469

The fth1-dp8 vector. The recombinant protein 8 gene of the fth1 vector (VIII-STS) was modified by introducing the 5′ (A – orange) and 3′ (B – blue) Illumina adaptors such that they flank the DNA insert (E). Reference DNA barcodes were introduced between the A adaptor and the first SfiI site (bc, see “ Pre-processing of sequence data” in Methods). Samples for deep sequencing are generated directly by PCR using the adaptors as PCR primers.
Figure Legend Snippet: The fth1-dp8 vector. The recombinant protein 8 gene of the fth1 vector (VIII-STS) was modified by introducing the 5′ (A – orange) and 3′ (B – blue) Illumina adaptors such that they flank the DNA insert (E). Reference DNA barcodes were introduced between the A adaptor and the first SfiI site (bc, see “ Pre-processing of sequence data” in Methods). Samples for deep sequencing are generated directly by PCR using the adaptors as PCR primers.

Techniques Used: Plasmid Preparation, Recombinant, Modification, Sequencing, Generated, Polymerase Chain Reaction

18) Product Images from "Discovery of Rare Mutations in Populations: TILLING by Sequencing 1Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C]Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C] [W]Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C] [W] [OA]"

Article Title: Discovery of Rare Mutations in Populations: TILLING by Sequencing 1Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C]Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C] [W]Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C] [W] [OA]

Journal: Plant Physiology

doi: 10.1104/pp.110.169748

Sequencing coverage and mutation discovery in tridimensionally pooled gene fragments. In this experiment (no. 3), target PCR products amplified from 44 template pools encompassing 64× or 48× combinations of 768 individuals were sequenced. A, The average sequencing depth in the 44 pools of each rice gene fragment is displayed. The boxed data sets correspond to the genes in B and C. B, Example of intragenic fluctuation in coverage associated with extreme GC contents. The top panel displays the percentage GC in a 10-bp window moved in 1-bp increments along the rice gene Os04g56580. The bottom panel reports the 0 to 5,000× coverage range. High GC content regions (arrows) correspond to low sequencing coverage. C, Inositol kinase-like (Os09g34300) gene of rice. The top panel displays sequencing coverage. The change frequencies are plotted versus the base position on the TILLED DNA fragment. In rice, GC > AT are induced most commonly, but not exclusively. G288A and C1319T were tested and confirmed to be heterozygous and homozygous, respectively. Numbers next to mutations are CAMBa F t scores, where higher values correspond to increased probability of true positive. The arrow points to a singleton signal (orphan). [See online article for color version of this figure.]
Figure Legend Snippet: Sequencing coverage and mutation discovery in tridimensionally pooled gene fragments. In this experiment (no. 3), target PCR products amplified from 44 template pools encompassing 64× or 48× combinations of 768 individuals were sequenced. A, The average sequencing depth in the 44 pools of each rice gene fragment is displayed. The boxed data sets correspond to the genes in B and C. B, Example of intragenic fluctuation in coverage associated with extreme GC contents. The top panel displays the percentage GC in a 10-bp window moved in 1-bp increments along the rice gene Os04g56580. The bottom panel reports the 0 to 5,000× coverage range. High GC content regions (arrows) correspond to low sequencing coverage. C, Inositol kinase-like (Os09g34300) gene of rice. The top panel displays sequencing coverage. The change frequencies are plotted versus the base position on the TILLED DNA fragment. In rice, GC > AT are induced most commonly, but not exclusively. G288A and C1319T were tested and confirmed to be heterozygous and homozygous, respectively. Numbers next to mutations are CAMBa F t scores, where higher values correspond to increased probability of true positive. The arrow points to a singleton signal (orphan). [See online article for color version of this figure.]

Techniques Used: Sequencing, Mutagenesis, Polymerase Chain Reaction, Amplification

Effect of template pooling and sequencing coverage on mutation detection and noise. DNA from rice individual heterozygous for three mutations ( RDR2 , A808G; PITPK , C1326T; TGT2A , T469C) were mixed with wild-type DNA to provide different dilutions of the mutated template. EMS mutagenesis in rice induces both conventional GC pair-to-AT pair changes (such as C1326T) as well as unconventional changes (such as A808G and T469C). The diluted templates were then used in triplicate PCR amplification of target genes followed by sequencing. Thus, the experiment entailed 12 libraries from PCR and sequencing of 3 genes × 4 dilutions × 3 replicates. A, Each of the three genes is represented in a separate panel that illustrates the per-base sequencing coverage in 12 libraries (top panels) and observed base change frequency (bottom panels). For example, the RDR2 track displays the frequency of A > G changes. Base positions with coverage below 1,000 are represented in red to highlight the contribution of low-coverage bases to noise. B, Effect of coverage on the noise of equally pooled mutations. The pooling factor is 1:95 heterozygous mutant:wild types. The change corresponding to the test mutation is indicated by red circles. C, Left, Detection of the three mutations at each pool dilution. The box covers the interquartile range of the distribution. The green/white circles correspond to the expected frequencies for dilutions of one heterozygous mutation in 63, 95, 127, and 191 wild-type individuals. Right, Sequencing error calls after stringent quality filtering. The rate for each base change type is represented by outlier box plots. The rate was collected from the pooled data set excluding changes in bases that were covered less than 1,000 times and changes more frequent than 0.4. The box represents the interquartile range and contains the mean confidence interval diamond. The individual dots are outlying points in the distribution defined by being farther from the edge of the box than 1.5× the interquartile range. The red brackets represent the shortest half (most dense 50% of observations). For each base pair set, the average error rate is shown on top.
Figure Legend Snippet: Effect of template pooling and sequencing coverage on mutation detection and noise. DNA from rice individual heterozygous for three mutations ( RDR2 , A808G; PITPK , C1326T; TGT2A , T469C) were mixed with wild-type DNA to provide different dilutions of the mutated template. EMS mutagenesis in rice induces both conventional GC pair-to-AT pair changes (such as C1326T) as well as unconventional changes (such as A808G and T469C). The diluted templates were then used in triplicate PCR amplification of target genes followed by sequencing. Thus, the experiment entailed 12 libraries from PCR and sequencing of 3 genes × 4 dilutions × 3 replicates. A, Each of the three genes is represented in a separate panel that illustrates the per-base sequencing coverage in 12 libraries (top panels) and observed base change frequency (bottom panels). For example, the RDR2 track displays the frequency of A > G changes. Base positions with coverage below 1,000 are represented in red to highlight the contribution of low-coverage bases to noise. B, Effect of coverage on the noise of equally pooled mutations. The pooling factor is 1:95 heterozygous mutant:wild types. The change corresponding to the test mutation is indicated by red circles. C, Left, Detection of the three mutations at each pool dilution. The box covers the interquartile range of the distribution. The green/white circles correspond to the expected frequencies for dilutions of one heterozygous mutation in 63, 95, 127, and 191 wild-type individuals. Right, Sequencing error calls after stringent quality filtering. The rate for each base change type is represented by outlier box plots. The rate was collected from the pooled data set excluding changes in bases that were covered less than 1,000 times and changes more frequent than 0.4. The box represents the interquartile range and contains the mean confidence interval diamond. The individual dots are outlying points in the distribution defined by being farther from the edge of the box than 1.5× the interquartile range. The red brackets represent the shortest half (most dense 50% of observations). For each base pair set, the average error rate is shown on top.

Techniques Used: Sequencing, Mutagenesis, Polymerase Chain Reaction, Amplification

19) Product Images from "The Microbiome of Ehrlichia-Infected and Uninfected Lone Star Ticks (Amblyomma americanum)"

Article Title: The Microbiome of Ehrlichia-Infected and Uninfected Lone Star Ticks (Amblyomma americanum)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0146651

Anaplasma , Borrelia , Coxiella , Ehrlichia , and Rickettsia were identified in Amblyomma americanum . Heat map shows relative abundance of OTUs of public health importance. It also reveals co-infections in 15 of the specimens, as well as additional Ehrlichia and Anaplasma infections missed by PCR amplification of groEL genes. Specimen = tick identification number; PCR = red are negative and green are positive via PCR amplification of groEL with Ehrlichia -specific primers. Sanger sequencing of groEL amplicons provided taxonomic information: AO = Anaplasma odoicoili , EC = Ehrlichia chaffeensis , EE = E . ewingii , PM = Panola Mountain Ehrlichia ). Sex = pink are female and blue are males.
Figure Legend Snippet: Anaplasma , Borrelia , Coxiella , Ehrlichia , and Rickettsia were identified in Amblyomma americanum . Heat map shows relative abundance of OTUs of public health importance. It also reveals co-infections in 15 of the specimens, as well as additional Ehrlichia and Anaplasma infections missed by PCR amplification of groEL genes. Specimen = tick identification number; PCR = red are negative and green are positive via PCR amplification of groEL with Ehrlichia -specific primers. Sanger sequencing of groEL amplicons provided taxonomic information: AO = Anaplasma odoicoili , EC = Ehrlichia chaffeensis , EE = E . ewingii , PM = Panola Mountain Ehrlichia ). Sex = pink are female and blue are males.

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

20) Product Images from "Identification of genes specifically or preferentially expressed in maize silk reveals similarity and diversity in transcript abundance of different dry stigmas"

Article Title: Identification of genes specifically or preferentially expressed in maize silk reveals similarity and diversity in transcript abundance of different dry stigmas

Journal: BMC Genomics

doi: 10.1186/1471-2164-13-294

Validation of RNA-seq results by real-time PCR. Transcript abundance represented by the two heat maps are the average of transcript abundance values from three independent real-time PCR experiments (left) and RPKM values of the RNA-seq analysis (middle). For each gene, the tissue with the maximum transcript abundance was set to 100, and relative transcript abundances in the other three tissues were calculated based on this maximum. Relative expression is represented by color scales as indicated (right). R is the correlation coefficient value between the two platforms.
Figure Legend Snippet: Validation of RNA-seq results by real-time PCR. Transcript abundance represented by the two heat maps are the average of transcript abundance values from three independent real-time PCR experiments (left) and RPKM values of the RNA-seq analysis (middle). For each gene, the tissue with the maximum transcript abundance was set to 100, and relative transcript abundances in the other three tissues were calculated based on this maximum. Relative expression is represented by color scales as indicated (right). R is the correlation coefficient value between the two platforms.

Techniques Used: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Expressing

21) Product Images from "Comparative Transcriptome Analyses Reveal Potential Mechanisms of Enhanced Drought Tolerance in Transgenic Salvia Miltiorrhiza Plants Expressing AtDREB1A from Arabidopsis"

Article Title: Comparative Transcriptome Analyses Reveal Potential Mechanisms of Enhanced Drought Tolerance in Transgenic Salvia Miltiorrhiza Plants Expressing AtDREB1A from Arabidopsis

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19030827

Correlations in changes in gene expression between fold-change determined from RNA-seq data (X-axis) and data obtained using qRT-PCR (Y-axis). WT, wild-type; pRD29A::AtDREB1A-31, pRD29A::AtDREB1A transgenic line 31. BD, before drought; AD, after six days of drought.
Figure Legend Snippet: Correlations in changes in gene expression between fold-change determined from RNA-seq data (X-axis) and data obtained using qRT-PCR (Y-axis). WT, wild-type; pRD29A::AtDREB1A-31, pRD29A::AtDREB1A transgenic line 31. BD, before drought; AD, after six days of drought.

Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Transgenic Assay

22) Product Images from "The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells"

Article Title: The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx537

Analyses of tRNA HisGUG and td-piR HisGUG in BmThg1l-depleted BmN4 cells. ( A ) Pie chart showing the 5′-terminal variations of BmN4 5′-half HisGUG identified by cP-RNA-seq. ( B ) Pie charts showing the 5′-terminal variations of BmN4 td-piR HisGUG bound to Siwi and BmAgo3. ( C ) BmThg1l mRNA from BmN4 cells treated with dsRNAs targeting Renilla luciferase (Rluc, negative control) or BmThg1l was quantified by qRT-PCR. Each data set represents the average of three independent experiments with bars showing the SD. ( D ) Total RNA from Rluc- or BmThg1l-depleted cells was subjected to denaturing PAGE and stained by SYBR Gold. Long exposure enabled clear observation of the piRNA bands. ( E, F ) Total RNA from Rluc- or BmThg1l-depleted cells was subjected to Northern blot targeting the 5′-part of tRNA HisGUG using a large-size gel with 1 nt resolution for mature tRNA (E) or a standard-size gel for its 5′-half (F). Because the 5′-part of the tRNA is targeted, both mature tRNA HisGUG and 5′-half HisGUG were detected. td-piR HisGUG was not detected due to a lack of sensitivity. For both mature tRNA and 5′-half, a band with slightly smaller size appeared upon BmThg1l-depletion, suggesting the role of BmThg1l in the –1 nucleotide addition to tRNA HisGUG . Bands shown with an asterisk might be non-specific as they seemed unaffected by the BmThg1l depletion. ( G ) The indicated piRNAs and let-7 miRNA in the Rluc- or BmThg1l-depleted cells were quantified by TaqMan qRT-PCR ( Supplementary Figure S1 ) or qRT-PCR using a stem-loop primer. The amounts in Rluc-depleted cells were set as 1, and relative amounts are indicated. Averages of three independent experiments with SD values are shown.
Figure Legend Snippet: Analyses of tRNA HisGUG and td-piR HisGUG in BmThg1l-depleted BmN4 cells. ( A ) Pie chart showing the 5′-terminal variations of BmN4 5′-half HisGUG identified by cP-RNA-seq. ( B ) Pie charts showing the 5′-terminal variations of BmN4 td-piR HisGUG bound to Siwi and BmAgo3. ( C ) BmThg1l mRNA from BmN4 cells treated with dsRNAs targeting Renilla luciferase (Rluc, negative control) or BmThg1l was quantified by qRT-PCR. Each data set represents the average of three independent experiments with bars showing the SD. ( D ) Total RNA from Rluc- or BmThg1l-depleted cells was subjected to denaturing PAGE and stained by SYBR Gold. Long exposure enabled clear observation of the piRNA bands. ( E, F ) Total RNA from Rluc- or BmThg1l-depleted cells was subjected to Northern blot targeting the 5′-part of tRNA HisGUG using a large-size gel with 1 nt resolution for mature tRNA (E) or a standard-size gel for its 5′-half (F). Because the 5′-part of the tRNA is targeted, both mature tRNA HisGUG and 5′-half HisGUG were detected. td-piR HisGUG was not detected due to a lack of sensitivity. For both mature tRNA and 5′-half, a band with slightly smaller size appeared upon BmThg1l-depletion, suggesting the role of BmThg1l in the –1 nucleotide addition to tRNA HisGUG . Bands shown with an asterisk might be non-specific as they seemed unaffected by the BmThg1l depletion. ( G ) The indicated piRNAs and let-7 miRNA in the Rluc- or BmThg1l-depleted cells were quantified by TaqMan qRT-PCR ( Supplementary Figure S1 ) or qRT-PCR using a stem-loop primer. The amounts in Rluc-depleted cells were set as 1, and relative amounts are indicated. Averages of three independent experiments with SD values are shown.

Techniques Used: RNA Sequencing Assay, Luciferase, Negative Control, Quantitative RT-PCR, Polyacrylamide Gel Electrophoresis, Staining, Northern Blot

Analyses of 5′-tRNA halves and td-piRNAs in BmNSun2-depleted BmN4 cells. ( A ) BmNSun2 mRNA from BmN4 cells treated with dsRNAs targeting Rluc (negative control) or BmNSun2 was quantified by qRT-PCR. Each data set represents the average of three independent experiments with bars showing the SD. ( B ) Total RNA from Rluc- or BmNSun2-depleted cells was subjected to denaturing PAGE and stained by SYBR Gold. Long exposure enabled clear observation of the piRNA bands. ( C ) Total RNA from Rluc- or BmNSun2-depleted cells was subjected to northern blot targeting the Bombyx 5S rRNA. ( D ) Total RNA from Rluc- or BmNSun2-depleted cells was subjected to Northern blot targeting the 5′-part of mature tRNA AspGUC and tRNA HisGUG . Because the 5′-part of the tRNA was targeted, 5′-half and td-piRNA, as well as mature tRNA, were all detected. We failed to detect td-piR HisGUG due to a lack of sensitivity. The northern blot bands were quantified and shown as relative abundance in the right graph. Abundances in Rluc-depleted cells were set as 1, and the averages of three independent experiments with bars showing the SD are shown. ( E ) The 5′-halves and td-piRNAs in Rluc- or BmNSun2-depleted cells were quantified by TaqMan qRT-PCR. The amounts in Rluc-depleted cells were set as 1, and relative amounts are indicated. Averages of three independent experiments with SD values are shown.
Figure Legend Snippet: Analyses of 5′-tRNA halves and td-piRNAs in BmNSun2-depleted BmN4 cells. ( A ) BmNSun2 mRNA from BmN4 cells treated with dsRNAs targeting Rluc (negative control) or BmNSun2 was quantified by qRT-PCR. Each data set represents the average of three independent experiments with bars showing the SD. ( B ) Total RNA from Rluc- or BmNSun2-depleted cells was subjected to denaturing PAGE and stained by SYBR Gold. Long exposure enabled clear observation of the piRNA bands. ( C ) Total RNA from Rluc- or BmNSun2-depleted cells was subjected to northern blot targeting the Bombyx 5S rRNA. ( D ) Total RNA from Rluc- or BmNSun2-depleted cells was subjected to Northern blot targeting the 5′-part of mature tRNA AspGUC and tRNA HisGUG . Because the 5′-part of the tRNA was targeted, 5′-half and td-piRNA, as well as mature tRNA, were all detected. We failed to detect td-piR HisGUG due to a lack of sensitivity. The northern blot bands were quantified and shown as relative abundance in the right graph. Abundances in Rluc-depleted cells were set as 1, and the averages of three independent experiments with bars showing the SD are shown. ( E ) The 5′-halves and td-piRNAs in Rluc- or BmNSun2-depleted cells were quantified by TaqMan qRT-PCR. The amounts in Rluc-depleted cells were set as 1, and relative amounts are indicated. Averages of three independent experiments with SD values are shown.

Techniques Used: Negative Control, Quantitative RT-PCR, Polyacrylamide Gel Electrophoresis, Staining, Northern Blot

Terminal structure analyses of tRNA halves expressed in BmN4 cells. ( A ) The 3′-terminal structures of 5′-tRNA halves and td-piRNAs derived from tRNA AspGUC and tRNA HisGUG were analyzed enzymatically. BmN4 total RNA was treated with CIP or T4 PNK (PNK). NT designates non-treated samples used as negative controls. The treated total RNA was subjected to 3′-adapter ligation by T4 Rnl, and the ligation efficiency was estimated by quantifying adapter-ligated target RNAs (5′-tRNA halves or piRNAs) using TaqMan qRT-PCR. Ligated products in NT samples were set as 1, and relative amounts are indicated. Averages of three independent experiments with SD values are shown. ( B ) To analyze the 3′-terminal structure of 3′-tRNA halves, BmN4 total RNA was subjected to deacylation treatment and sodium periodate oxidation, followed by β-elimination. The mature tRNA AspGUC , 3′-half AspGUC , tRNA HisGUG , 3′-half HisGUG , and let-7 miRNA (control) in the treated total RNA were analyzed by Northern blots.
Figure Legend Snippet: Terminal structure analyses of tRNA halves expressed in BmN4 cells. ( A ) The 3′-terminal structures of 5′-tRNA halves and td-piRNAs derived from tRNA AspGUC and tRNA HisGUG were analyzed enzymatically. BmN4 total RNA was treated with CIP or T4 PNK (PNK). NT designates non-treated samples used as negative controls. The treated total RNA was subjected to 3′-adapter ligation by T4 Rnl, and the ligation efficiency was estimated by quantifying adapter-ligated target RNAs (5′-tRNA halves or piRNAs) using TaqMan qRT-PCR. Ligated products in NT samples were set as 1, and relative amounts are indicated. Averages of three independent experiments with SD values are shown. ( B ) To analyze the 3′-terminal structure of 3′-tRNA halves, BmN4 total RNA was subjected to deacylation treatment and sodium periodate oxidation, followed by β-elimination. The mature tRNA AspGUC , 3′-half AspGUC , tRNA HisGUG , 3′-half HisGUG , and let-7 miRNA (control) in the treated total RNA were analyzed by Northern blots.

Techniques Used: Derivative Assay, Ligation, Quantitative RT-PCR, Northern Blot

23) Product Images from "Development of Personalized Tumor Biomarkers Using Massively Parallel Sequencing"

Article Title: Development of Personalized Tumor Biomarkers Using Massively Parallel Sequencing

Journal: Science translational medicine

doi: 10.1126/scitranslmed.3000702

Detection of tumor-specific rearrangements in mixtures of tumor and normal DNA. Decreasing amounts of tumor DNA were mixed with increasing amounts of normal tissue DNA (300 ng total) and were used as template molecules for PCR using chromosome 4:8 translocation-specific primers or chromosome 3 control primers (see Materials and Methods for additional information).
Figure Legend Snippet: Detection of tumor-specific rearrangements in mixtures of tumor and normal DNA. Decreasing amounts of tumor DNA were mixed with increasing amounts of normal tissue DNA (300 ng total) and were used as template molecules for PCR using chromosome 4:8 translocation-specific primers or chromosome 3 control primers (see Materials and Methods for additional information).

Techniques Used: Polymerase Chain Reaction, Translocation Assay

Detection of tumor-specific rearrangements in plasma of cancer patients. (A) The identified chromosome 4:8 and 16 rearrangements were used to design PCR primers spanning breakpoints and to amplify rearranged DNA from tumor tissue and plasma from patients H×402 and H×403, respectively. A plasma sample from an unrelated healthy individual was used as a control for both rearrangements. (B) Plasma samples from patient H×402 were analyzed at different time points using digital PCR to determine the fraction of genomic equivalents of plasma DNA containing the chromosome 4:8 rearrangement. The fraction of rearranged DNA at day 137 was 0.3%, consistent with residual metastatic lesions present in the remaining lobe of the liver.
Figure Legend Snippet: Detection of tumor-specific rearrangements in plasma of cancer patients. (A) The identified chromosome 4:8 and 16 rearrangements were used to design PCR primers spanning breakpoints and to amplify rearranged DNA from tumor tissue and plasma from patients H×402 and H×403, respectively. A plasma sample from an unrelated healthy individual was used as a control for both rearrangements. (B) Plasma samples from patient H×402 were analyzed at different time points using digital PCR to determine the fraction of genomic equivalents of plasma DNA containing the chromosome 4:8 rearrangement. The fraction of rearranged DNA at day 137 was 0.3%, consistent with residual metastatic lesions present in the remaining lobe of the liver.

Techniques Used: Polymerase Chain Reaction, Digital PCR

24) Product Images from "Circular RNA CRIM1 functions as a ceRNA to promote nasopharyngeal carcinoma metastasis and docetaxel chemoresistance through upregulating FOXQ1"

Article Title: Circular RNA CRIM1 functions as a ceRNA to promote nasopharyngeal carcinoma metastasis and docetaxel chemoresistance through upregulating FOXQ1

Journal: Molecular Cancer

doi: 10.1186/s12943-020-01149-x

CircCRIM1 is upregulated in NPC patients with distant metastasis and promotes NPC cell metastasis and EMT in vitro. a Volcano plots of differential circRNAs between high metastasis S18 cells and low metastasis S26 cells. b Genomic loci of the circCRIM1 gene. The red and green arrows represent divergent primers. The backsplicing junction was validated by Sanger sequencing. c Quantitative real-time PCR (qRT-PCR) analysis of GAPDH, CRIM1 mRNA and circCRIM1 expression after RNase R treatment. d circCRIM1 expression in cDNA and genomic DNA (gDNA). GAPDH was used as a negative control. e Relative expression of circCRIM1 in S18, HONE1 and S26 cells. f Relative expression of circCRIM1 in NPC tissue without ( n = 157) or with ( n = 61) distant metastasis. g-h Representative and quantified results of the Transwell migration and invasion assays in NPC cells transfected with circCRIM1 siRNA or scrambled control (siSCR, g ) or with circCRIM1 overexpression plasmid or vector ( h ). Scale bar, 100 μm. i Immunofluorescence images of E-cadherin and Vimentin expression in NPC cells with or without circCRIM1 downregulation. Scale bar, 10 μm. j Western blotting analysis of E-cadherin, N-cadherin and Vimentin in NPC cells with or without circCRIM1 depletion. The data are presented as the mean ± S.D. All in vitro data are representative of three independent experiments. Student’s t -test; * P
Figure Legend Snippet: CircCRIM1 is upregulated in NPC patients with distant metastasis and promotes NPC cell metastasis and EMT in vitro. a Volcano plots of differential circRNAs between high metastasis S18 cells and low metastasis S26 cells. b Genomic loci of the circCRIM1 gene. The red and green arrows represent divergent primers. The backsplicing junction was validated by Sanger sequencing. c Quantitative real-time PCR (qRT-PCR) analysis of GAPDH, CRIM1 mRNA and circCRIM1 expression after RNase R treatment. d circCRIM1 expression in cDNA and genomic DNA (gDNA). GAPDH was used as a negative control. e Relative expression of circCRIM1 in S18, HONE1 and S26 cells. f Relative expression of circCRIM1 in NPC tissue without ( n = 157) or with ( n = 61) distant metastasis. g-h Representative and quantified results of the Transwell migration and invasion assays in NPC cells transfected with circCRIM1 siRNA or scrambled control (siSCR, g ) or with circCRIM1 overexpression plasmid or vector ( h ). Scale bar, 100 μm. i Immunofluorescence images of E-cadherin and Vimentin expression in NPC cells with or without circCRIM1 downregulation. Scale bar, 10 μm. j Western blotting analysis of E-cadherin, N-cadherin and Vimentin in NPC cells with or without circCRIM1 depletion. The data are presented as the mean ± S.D. All in vitro data are representative of three independent experiments. Student’s t -test; * P

Techniques Used: In Vitro, Sequencing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Negative Control, Migration, Transfection, Over Expression, Plasmid Preparation, Immunofluorescence, Western Blot

25) Product Images from "Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing"

Article Title: Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing

Journal: Genome Biology

doi: 10.1186/s13059-018-1518-x

Validation of a reporter-less NHEJ assay in cells deficient for XRCC4 . a Deletion of the intervening sequence between two target sites by paired Cas9-gRNA. Cells were transfected with expression plasmids for single or paired sgRNAs and Cas9 and genomic DNA was purified 72 h post-transfection and amplified by primers flanking the cutting sites. The PCR amplicons were subjected to agarose gel electrophoresis. The distance between two cleavage sites was 57 bp at the LDHA site and 33 bp at the ROSA26 site as indicated and the deletion of the intervening sequence indicates simultaneous Cas9 cleavage. b–d Analysis of NHEJ induced by paired Cas9-gRNA at the LDHA and ROSA26 sites in isogenic XRCC4 +/+ and XRCC4 −/− mouse ES cells. The normalized editing efficiency ( b ), the frequency of each group in edited events ( c ), and the frequency of each category in group I events ( d ) were calculated. The normalized editing efficiency represents the efficiency of overall NHEJ, including accurate and mutagenic NHEJ. Bars represent the mean ± standard deviation (SD) of three independent experiments. For normalized editing efficiency ( b ), Student’s paired t -test between XRCC4 +/+ and XRCC4 −/− P = 0.0028 at the LDHA site. e Deletion length distributions of Group I ‘Del’ events in isogenic XRCC4 +/+ and XRCC4 −/− mouse ES cells. The reads were combined by three independent experiments. At the LDHA site and the ROSA26 site, each dot represents 100 reads and 20 reads, respectively. The median deletion length is indicated, and deletion distributions demonstrate a shift towards longer deletions in cells lacking XRCC4 (Mann–Whitney test, **** P
Figure Legend Snippet: Validation of a reporter-less NHEJ assay in cells deficient for XRCC4 . a Deletion of the intervening sequence between two target sites by paired Cas9-gRNA. Cells were transfected with expression plasmids for single or paired sgRNAs and Cas9 and genomic DNA was purified 72 h post-transfection and amplified by primers flanking the cutting sites. The PCR amplicons were subjected to agarose gel electrophoresis. The distance between two cleavage sites was 57 bp at the LDHA site and 33 bp at the ROSA26 site as indicated and the deletion of the intervening sequence indicates simultaneous Cas9 cleavage. b–d Analysis of NHEJ induced by paired Cas9-gRNA at the LDHA and ROSA26 sites in isogenic XRCC4 +/+ and XRCC4 −/− mouse ES cells. The normalized editing efficiency ( b ), the frequency of each group in edited events ( c ), and the frequency of each category in group I events ( d ) were calculated. The normalized editing efficiency represents the efficiency of overall NHEJ, including accurate and mutagenic NHEJ. Bars represent the mean ± standard deviation (SD) of three independent experiments. For normalized editing efficiency ( b ), Student’s paired t -test between XRCC4 +/+ and XRCC4 −/− P = 0.0028 at the LDHA site. e Deletion length distributions of Group I ‘Del’ events in isogenic XRCC4 +/+ and XRCC4 −/− mouse ES cells. The reads were combined by three independent experiments. At the LDHA site and the ROSA26 site, each dot represents 100 reads and 20 reads, respectively. The median deletion length is indicated, and deletion distributions demonstrate a shift towards longer deletions in cells lacking XRCC4 (Mann–Whitney test, **** P

Techniques Used: Non-Homologous End Joining, Sequencing, Transfection, Expressing, Purification, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Standard Deviation, MANN-WHITNEY

26) Product Images from "Deep sustained response to daratumumab monotherapy associated with T-cell expansion in triple refractory myeloma"

Article Title: Deep sustained response to daratumumab monotherapy associated with T-cell expansion in triple refractory myeloma

Journal: Experimental Hematology & Oncology

doi: 10.1186/s40164-018-0096-7

Change in immune cell populations and properties. A diagram of the T-cell receptor sequencing protocol is shown in a . DNA was extracted from peripheral blood mononuclear cells and subjected to 2 rounds of PCR amplification. During the first round, primers specific for the variable (forward) and joining (reverse) were used to amplify somatically recombined T-cell receptors. During the second round, each amplicon was amplified using primers containing barcode and adapter sequences to facilitate T-cell receptor sequencing via the Illumina platform in the next step of the protocol. The sequencing data were used to evaluate T-cell receptor clonality, diversity, and changes from baseline. Percent changes in CD8 T cells from baseline among patients in the SIRIUS study are shown in b . The patient, indicated by the red line, showed a rapid expansion of CD8 + T cells that was maintained over time. c The percent change from baseline of regulatory T cells over time. d T-cell receptor clonality at baseline versus on-treatment time points. For all patients in black, the on-treatment time point was 3 months. For the patient, the sample time points were 3 and 32 months, in orange and red, respectively. e The T-cell receptor clonality in the case study patient at baseline, 3, and 32 months. PBMCs peripheral blood mononuclear cells, V variable region, D diversity region, J joining region, PCR polymerase chain reaction, TCR T-cell receptor
Figure Legend Snippet: Change in immune cell populations and properties. A diagram of the T-cell receptor sequencing protocol is shown in a . DNA was extracted from peripheral blood mononuclear cells and subjected to 2 rounds of PCR amplification. During the first round, primers specific for the variable (forward) and joining (reverse) were used to amplify somatically recombined T-cell receptors. During the second round, each amplicon was amplified using primers containing barcode and adapter sequences to facilitate T-cell receptor sequencing via the Illumina platform in the next step of the protocol. The sequencing data were used to evaluate T-cell receptor clonality, diversity, and changes from baseline. Percent changes in CD8 T cells from baseline among patients in the SIRIUS study are shown in b . The patient, indicated by the red line, showed a rapid expansion of CD8 + T cells that was maintained over time. c The percent change from baseline of regulatory T cells over time. d T-cell receptor clonality at baseline versus on-treatment time points. For all patients in black, the on-treatment time point was 3 months. For the patient, the sample time points were 3 and 32 months, in orange and red, respectively. e The T-cell receptor clonality in the case study patient at baseline, 3, and 32 months. PBMCs peripheral blood mononuclear cells, V variable region, D diversity region, J joining region, PCR polymerase chain reaction, TCR T-cell receptor

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification

27) Product Images from "Discovery of Rare Mutations in Populations: TILLING by Sequencing 1Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C]Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C] [W]Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C] [W] [OA]"

Article Title: Discovery of Rare Mutations in Populations: TILLING by Sequencing 1Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C]Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C] [W]Discovery of Rare Mutations in Populations: TILLING by Sequencing 1 [C] [W] [OA]

Journal: Plant Physiology

doi: 10.1104/pp.110.169748

Sequencing coverage and mutation discovery in tridimensionally pooled gene fragments. In this experiment (no. 3), target PCR products amplified from 44 template pools encompassing 64× or 48× combinations of 768 individuals were sequenced. A, The average sequencing depth in the 44 pools of each rice gene fragment is displayed. The boxed data sets correspond to the genes in B and C. B, Example of intragenic fluctuation in coverage associated with extreme GC contents. The top panel displays the percentage GC in a 10-bp window moved in 1-bp increments along the rice gene Os04g56580. The bottom panel reports the 0 to 5,000× coverage range. High GC content regions (arrows) correspond to low sequencing coverage. C, Inositol kinase-like (Os09g34300) gene of rice. The top panel displays sequencing coverage. The change frequencies are plotted versus the base position on the TILLED DNA fragment. In rice, GC > AT are induced most commonly, but not exclusively. G288A and C1319T were tested and confirmed to be heterozygous and homozygous, respectively. Numbers next to mutations are CAMBa F t scores, where higher values correspond to increased probability of true positive. The arrow points to a singleton signal (orphan). [See online article for color version of this figure.]
Figure Legend Snippet: Sequencing coverage and mutation discovery in tridimensionally pooled gene fragments. In this experiment (no. 3), target PCR products amplified from 44 template pools encompassing 64× or 48× combinations of 768 individuals were sequenced. A, The average sequencing depth in the 44 pools of each rice gene fragment is displayed. The boxed data sets correspond to the genes in B and C. B, Example of intragenic fluctuation in coverage associated with extreme GC contents. The top panel displays the percentage GC in a 10-bp window moved in 1-bp increments along the rice gene Os04g56580. The bottom panel reports the 0 to 5,000× coverage range. High GC content regions (arrows) correspond to low sequencing coverage. C, Inositol kinase-like (Os09g34300) gene of rice. The top panel displays sequencing coverage. The change frequencies are plotted versus the base position on the TILLED DNA fragment. In rice, GC > AT are induced most commonly, but not exclusively. G288A and C1319T were tested and confirmed to be heterozygous and homozygous, respectively. Numbers next to mutations are CAMBa F t scores, where higher values correspond to increased probability of true positive. The arrow points to a singleton signal (orphan). [See online article for color version of this figure.]

Techniques Used: Sequencing, Mutagenesis, Polymerase Chain Reaction, Amplification

Effect of template pooling and sequencing coverage on mutation detection and noise. DNA from rice individual heterozygous for three mutations ( RDR2 , A808G; PITPK , C1326T; TGT2A , T469C) were mixed with wild-type DNA to provide different dilutions of the mutated template. EMS mutagenesis in rice induces both conventional GC pair-to-AT pair changes (such as C1326T) as well as unconventional changes (such as A808G and T469C). The diluted templates were then used in triplicate PCR amplification of target genes followed by sequencing. Thus, the experiment entailed 12 libraries from PCR and sequencing of 3 genes × 4 dilutions × 3 replicates. A, Each of the three genes is represented in a separate panel that illustrates the per-base sequencing coverage in 12 libraries (top panels) and observed base change frequency (bottom panels). For example, the RDR2 track displays the frequency of A > G changes. Base positions with coverage below 1,000 are represented in red to highlight the contribution of low-coverage bases to noise. B, Effect of coverage on the noise of equally pooled mutations. The pooling factor is 1:95 heterozygous mutant:wild types. The change corresponding to the test mutation is indicated by red circles. C, Left, Detection of the three mutations at each pool dilution. The box covers the interquartile range of the distribution. The green/white circles correspond to the expected frequencies for dilutions of one heterozygous mutation in 63, 95, 127, and 191 wild-type individuals. Right, Sequencing error calls after stringent quality filtering. The rate for each base change type is represented by outlier box plots. The rate was collected from the pooled data set excluding changes in bases that were covered less than 1,000 times and changes more frequent than 0.4. The box represents the interquartile range and contains the mean confidence interval diamond. The individual dots are outlying points in the distribution defined by being farther from the edge of the box than 1.5× the interquartile range. The red brackets represent the shortest half (most dense 50% of observations). For each base pair set, the average error rate is shown on top.
Figure Legend Snippet: Effect of template pooling and sequencing coverage on mutation detection and noise. DNA from rice individual heterozygous for three mutations ( RDR2 , A808G; PITPK , C1326T; TGT2A , T469C) were mixed with wild-type DNA to provide different dilutions of the mutated template. EMS mutagenesis in rice induces both conventional GC pair-to-AT pair changes (such as C1326T) as well as unconventional changes (such as A808G and T469C). The diluted templates were then used in triplicate PCR amplification of target genes followed by sequencing. Thus, the experiment entailed 12 libraries from PCR and sequencing of 3 genes × 4 dilutions × 3 replicates. A, Each of the three genes is represented in a separate panel that illustrates the per-base sequencing coverage in 12 libraries (top panels) and observed base change frequency (bottom panels). For example, the RDR2 track displays the frequency of A > G changes. Base positions with coverage below 1,000 are represented in red to highlight the contribution of low-coverage bases to noise. B, Effect of coverage on the noise of equally pooled mutations. The pooling factor is 1:95 heterozygous mutant:wild types. The change corresponding to the test mutation is indicated by red circles. C, Left, Detection of the three mutations at each pool dilution. The box covers the interquartile range of the distribution. The green/white circles correspond to the expected frequencies for dilutions of one heterozygous mutation in 63, 95, 127, and 191 wild-type individuals. Right, Sequencing error calls after stringent quality filtering. The rate for each base change type is represented by outlier box plots. The rate was collected from the pooled data set excluding changes in bases that were covered less than 1,000 times and changes more frequent than 0.4. The box represents the interquartile range and contains the mean confidence interval diamond. The individual dots are outlying points in the distribution defined by being farther from the edge of the box than 1.5× the interquartile range. The red brackets represent the shortest half (most dense 50% of observations). For each base pair set, the average error rate is shown on top.

Techniques Used: Sequencing, Mutagenesis, Polymerase Chain Reaction, Amplification

28) Product Images from "The BisPCR2 method for targeted bisulfite sequencing"

Article Title: The BisPCR2 method for targeted bisulfite sequencing

Journal: Epigenetics & Chromatin

doi: 10.1186/s13072-015-0020-x

BisPCR 2 DNA sequencing libraries. a Bioanalyzer gel visualizing the five amplicon fragments of a representative sample, ND1, following PCR#2. b Bioanalyzer electropherogram quantifying the amount of each fragment in ND1, illustrating the roughly equivalent amounts of all five amplicons. c Average reads per amplicon for each sample. ND non-diabetic, T2D type 2 diabetic.
Figure Legend Snippet: BisPCR 2 DNA sequencing libraries. a Bioanalyzer gel visualizing the five amplicon fragments of a representative sample, ND1, following PCR#2. b Bioanalyzer electropherogram quantifying the amount of each fragment in ND1, illustrating the roughly equivalent amounts of all five amplicons. c Average reads per amplicon for each sample. ND non-diabetic, T2D type 2 diabetic.

Techniques Used: DNA Sequencing, Amplification, Polymerase Chain Reaction

Schema of BisPCR 2 method for targeted bisulfite sequencing. DNA sequencing libraries are prepared by bisulfite conversion of genomic DNA followed by two rounds of PCR for target enrichment (PCR#1) and subsequent sample barcoding (PCR#2). Partial adapter overhangs are added to target enrichment primers to permit simplified library preparation by PCR. PCR#1 amplicons are pooled prior to the PCR#2 reaction for each biological sample. Due to the presence of the unique barcodes, all PCR#2 amplicons can be pooled for a single next-generation sequencing run.
Figure Legend Snippet: Schema of BisPCR 2 method for targeted bisulfite sequencing. DNA sequencing libraries are prepared by bisulfite conversion of genomic DNA followed by two rounds of PCR for target enrichment (PCR#1) and subsequent sample barcoding (PCR#2). Partial adapter overhangs are added to target enrichment primers to permit simplified library preparation by PCR. PCR#1 amplicons are pooled prior to the PCR#2 reaction for each biological sample. Due to the presence of the unique barcodes, all PCR#2 amplicons can be pooled for a single next-generation sequencing run.

Techniques Used: Methylation Sequencing, DNA Sequencing, Polymerase Chain Reaction, Next-Generation Sequencing

29) Product Images from "Improved workflows for high throughput library preparation using the transposome-based nextera system"

Article Title: Improved workflows for high throughput library preparation using the transposome-based nextera system

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-13-104

Library QC Tape station electropherogram. Nextera Post-PCR libraries constructed with a range of concentrations (post-normalisation) and gDNA from four different genomes: Mycobacterium tuberculosis (purple), Escherichia coli (blue), Clostridium difficile (red) and Plasmodium falciparum (green). Libraries were constructed using the standard Nextera protocol (A) . Evaluation of the Axyprep Mag Normaliser kit (B) : two individual C. difficile Nextera libraries were constructed using the standard Illumina protocol (light/dark green) and two with our normalisation workflow (light/dark purple). Where the standard library had very short inserts, our method produced a library with the normal size distribution. Evaluation of C. difficile Nextera Post-PCR libraries constructed using varying volume Nextera reactions (C) : standard (purple), half-volume (green), quarter-volume (red) and one-eighth volume (blue). Size distribution profiles of libraries constructed using normalisation followed by reaction E (D) : M. tuberculosis (purple), E. coli (blue), C. difficile (red) and P. falciparum (green).
Figure Legend Snippet: Library QC Tape station electropherogram. Nextera Post-PCR libraries constructed with a range of concentrations (post-normalisation) and gDNA from four different genomes: Mycobacterium tuberculosis (purple), Escherichia coli (blue), Clostridium difficile (red) and Plasmodium falciparum (green). Libraries were constructed using the standard Nextera protocol (A) . Evaluation of the Axyprep Mag Normaliser kit (B) : two individual C. difficile Nextera libraries were constructed using the standard Illumina protocol (light/dark green) and two with our normalisation workflow (light/dark purple). Where the standard library had very short inserts, our method produced a library with the normal size distribution. Evaluation of C. difficile Nextera Post-PCR libraries constructed using varying volume Nextera reactions (C) : standard (purple), half-volume (green), quarter-volume (red) and one-eighth volume (blue). Size distribution profiles of libraries constructed using normalisation followed by reaction E (D) : M. tuberculosis (purple), E. coli (blue), C. difficile (red) and P. falciparum (green).

Techniques Used: Polymerase Chain Reaction, Construct, Produced

30) Product Images from "Targeted sequencing library preparation by genomic DNA circularization"

Article Title: Targeted sequencing library preparation by genomic DNA circularization

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-11-122

Targeted sequencing library preparation method . (a) Overview of the assay. (b) Specific preparation steps: (1) genomic DNA is digested using Mse I restriction endonuclease. (2) Then, genomic DNA fragments are circularized using thermostable DNA ligase and Taq DNA polymerase for 5' editing. Pool of oligonucleotides targeting 5' and 3' ends of the DNA fragments and vector oligonucleotide are used for targeted DNA capture. (3) After circularization, regular Illumina sequencing library can be prepared by PCR. (4) PCR amplified library fragments are similar to regular Illumina library constructs and anneal to immobilized primers on the flow cell. (5) Additionally, circular constructs can be directly sequenced as the adapted genomic DNA circles incorporate all DNA components required for library immobilization and sequencing. (c) Molecular structures of vector oligonucleotide and capture oligonucleotides.
Figure Legend Snippet: Targeted sequencing library preparation method . (a) Overview of the assay. (b) Specific preparation steps: (1) genomic DNA is digested using Mse I restriction endonuclease. (2) Then, genomic DNA fragments are circularized using thermostable DNA ligase and Taq DNA polymerase for 5' editing. Pool of oligonucleotides targeting 5' and 3' ends of the DNA fragments and vector oligonucleotide are used for targeted DNA capture. (3) After circularization, regular Illumina sequencing library can be prepared by PCR. (4) PCR amplified library fragments are similar to regular Illumina library constructs and anneal to immobilized primers on the flow cell. (5) Additionally, circular constructs can be directly sequenced as the adapted genomic DNA circles incorporate all DNA components required for library immobilization and sequencing. (c) Molecular structures of vector oligonucleotide and capture oligonucleotides.

Techniques Used: Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Construct, Flow Cytometry

31) Product Images from "Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters"

Article Title: Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.00830

Detection of serially diluted virus samples by microfluidic nested PCR followed by MiSeq amplicon sequencing. Plasmid DNA and cDNA samples containing target regions of each virus were mixed at equimolar concentrations and diluted to 10 4 –10 0 copies/μL. The mixtures were subjected to the first round of MF-PCR, and the amplicon was collected and used for MFnPCR with 20 oligonucleotide primer sets for detection of 11 viruses (i.e., AiV; AsV; EV; NoV GI, GII, and GIV; HAV; HEV; RV; SaV; and HPV). C T values determined in the nested MF PCR are shown as a heat map. Amplicon size represents the size of a PCR amplicon without an Illumina tag sequence.
Figure Legend Snippet: Detection of serially diluted virus samples by microfluidic nested PCR followed by MiSeq amplicon sequencing. Plasmid DNA and cDNA samples containing target regions of each virus were mixed at equimolar concentrations and diluted to 10 4 –10 0 copies/μL. The mixtures were subjected to the first round of MF-PCR, and the amplicon was collected and used for MFnPCR with 20 oligonucleotide primer sets for detection of 11 viruses (i.e., AiV; AsV; EV; NoV GI, GII, and GIV; HAV; HEV; RV; SaV; and HPV). C T values determined in the nested MF PCR are shown as a heat map. Amplicon size represents the size of a PCR amplicon without an Illumina tag sequence.

Techniques Used: Nested PCR, Amplification, Sequencing, Plasmid Preparation, Polymerase Chain Reaction

32) Product Images from "Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment"

Article Title: Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw650

Multiplexed NaME-PrO combined with targeted re-sequencing. Multiplexed-NaME-PrO applied to cfDNA from normal volunteers spiked with fragmented genomic DNA to generate ∼0.5% mutation abundance at multiple clinically relevant sites. Following NaME-PrO, samples were PCR-amplified and processed for library preparation. Variant to noise plots derived from Illumina MiSeq sequencing data are depicted. ( A and B ) KRAS G12V, G12R, G13D mutations in fragmented genomic DNA with and without application of NaME-PrO, respectively. ( C and D ) IDH1 R132C mutation in cfDNA with and without NaME-PrO, respectively. The original mutation abundance is 0.5% in both cases, while the read depth is about 1000–2000× depending on sequence. Inserts in B and D represent magnified versions of the same graphs to depict clearly the effect of sequencing noise. Application of NaME-PrO increases the signal-to-noise ratio and reveals clearly the rare mutation. Experiments were repeated two independent times.
Figure Legend Snippet: Multiplexed NaME-PrO combined with targeted re-sequencing. Multiplexed-NaME-PrO applied to cfDNA from normal volunteers spiked with fragmented genomic DNA to generate ∼0.5% mutation abundance at multiple clinically relevant sites. Following NaME-PrO, samples were PCR-amplified and processed for library preparation. Variant to noise plots derived from Illumina MiSeq sequencing data are depicted. ( A and B ) KRAS G12V, G12R, G13D mutations in fragmented genomic DNA with and without application of NaME-PrO, respectively. ( C and D ) IDH1 R132C mutation in cfDNA with and without NaME-PrO, respectively. The original mutation abundance is 0.5% in both cases, while the read depth is about 1000–2000× depending on sequence. Inserts in B and D represent magnified versions of the same graphs to depict clearly the effect of sequencing noise. Application of NaME-PrO increases the signal-to-noise ratio and reveals clearly the rare mutation. Experiments were repeated two independent times.

Techniques Used: Sequencing, Mutagenesis, Polymerase Chain Reaction, Amplification, Variant Assay, Derivative Assay

33) Product Images from "Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain"

Article Title: Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain

Journal: BMC Genomics

doi: 10.1186/1471-2164-12-518

Reproducibility of AEI assays ( GNB1L ) . (a) [Left] Comparison of the results of independent measurements of GNB1L AEI. (Each assay was performed independently, from the isolation of brain RNA through DNA sequencing.); [Right] Regression analysis. (b) Histogram of the distribution of r 2 values obtained in regression analysis of independent AEI assays for 40 candidate genes for which replica assays were carried out. (All data are shown). (c) [Left] Comparison of the results of independent measurements of GNB1L AEI using PCR/DNA sequencing-based (top) and PCR/SNaPShot ® -based (bottom) assays. [Right] Regression analysis. [Note: the Illumina-2 AEI ratios in (a) were matched with those obtained in the Illumina-1 AEI assay, which contained fewer samples. The full set of data from the Illumina-2 assay is shown in (c).]
Figure Legend Snippet: Reproducibility of AEI assays ( GNB1L ) . (a) [Left] Comparison of the results of independent measurements of GNB1L AEI. (Each assay was performed independently, from the isolation of brain RNA through DNA sequencing.); [Right] Regression analysis. (b) Histogram of the distribution of r 2 values obtained in regression analysis of independent AEI assays for 40 candidate genes for which replica assays were carried out. (All data are shown). (c) [Left] Comparison of the results of independent measurements of GNB1L AEI using PCR/DNA sequencing-based (top) and PCR/SNaPShot ® -based (bottom) assays. [Right] Regression analysis. [Note: the Illumina-2 AEI ratios in (a) were matched with those obtained in the Illumina-1 AEI assay, which contained fewer samples. The full set of data from the Illumina-2 assay is shown in (c).]

Techniques Used: Isolation, DNA Sequencing, Polymerase Chain Reaction

34) Product Images from "An Improved Protocol for Sequencing of Repetitive Genomic Regions and Structural Variations Using Mutagenesis and Next Generation Sequencing"

Article Title: An Improved Protocol for Sequencing of Repetitive Genomic Regions and Structural Variations Using Mutagenesis and Next Generation Sequencing

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043359

Overview of the simulated NG-SAM protocol. The numbering corresponds to the steps enumerated above in the main text. The trapezoids shaded in light blue represent PCR amplifications (with – being the number of cycles), while the rectangles shaded in yellow represent sampling of molecules by dilution. – are the number of molecules present in the various stages of the simulated experiment, with unique variants symbolised by different coloured dots. and are the dilution factors corresponding to the first and second dilution steps. The black lines represent the “lineages
Figure Legend Snippet: Overview of the simulated NG-SAM protocol. The numbering corresponds to the steps enumerated above in the main text. The trapezoids shaded in light blue represent PCR amplifications (with – being the number of cycles), while the rectangles shaded in yellow represent sampling of molecules by dilution. – are the number of molecules present in the various stages of the simulated experiment, with unique variants symbolised by different coloured dots. and are the dilution factors corresponding to the first and second dilution steps. The black lines represent the “lineages" of the molecules sampled by the second dilution, traced back to the initial molecule pool of size . The steps A – C correspond to the mutagenic PCR, dilution and cleanup PCR steps of the mutagenic protocol. simNGS [35] is a software for simulating Illumina sequencing and Velvet [8] is a short read assembler.

Techniques Used: Polymerase Chain Reaction, Sampling, Software, Sequencing

35) Product Images from "Transcriptomic Changes Due to Cytoplasmic TDP-43 Expression Reveal Dysregulation of Histone Transcripts and Nuclear Chromatin"

Article Title: Transcriptomic Changes Due to Cytoplasmic TDP-43 Expression Reveal Dysregulation of Histone Transcripts and Nuclear Chromatin

Journal: PLoS ONE

doi: 10.1371/journal.pone.0141836

Histone transcript dysregulation in ΔNLS-hTDP-43 mice. (A), Comparison of multimapped read fold change in canonical and variant histones shows a significant upregulation in canonical histones (fold change = 2.12, 2-sided t-test p = 0.046) and significant but slight downregulation in variant histones (fold change = 0.841, 2-sided t-test p = 0.017) in transgenic vs nontransgenic mice. (B), Analysis of log 2 (fold changes) of genes involved in histone 3’ UTR processing reveal an enrichment of significantly changing genes. (C), Analysis of TDP-43 CLIP binding on genes involved in histone 3’ UTR processing shows enrichment for CLIP binding. (D), RT-qPCR validation of histone 3’ UTR processing genes and canonical histones with an oligo-dT selection for polyadenylated RNA confirms the upregulation in aberrantly polyadenylated histone genes in the ΔNLS-TDP-43-expressing mice. (E), Similar RT-qPCR validation with random hexamer priming shows that non-polyadenylated canonical histones are not significantly upregulated in the bigenic mice. (F), Comparison of RNA-seq and RT-PCR analysis validates RNA-seq results, except for histone transcripts (white filled symbols) which revealed distinct results when priming with random hexamers versus oligo-dT’s. *p
Figure Legend Snippet: Histone transcript dysregulation in ΔNLS-hTDP-43 mice. (A), Comparison of multimapped read fold change in canonical and variant histones shows a significant upregulation in canonical histones (fold change = 2.12, 2-sided t-test p = 0.046) and significant but slight downregulation in variant histones (fold change = 0.841, 2-sided t-test p = 0.017) in transgenic vs nontransgenic mice. (B), Analysis of log 2 (fold changes) of genes involved in histone 3’ UTR processing reveal an enrichment of significantly changing genes. (C), Analysis of TDP-43 CLIP binding on genes involved in histone 3’ UTR processing shows enrichment for CLIP binding. (D), RT-qPCR validation of histone 3’ UTR processing genes and canonical histones with an oligo-dT selection for polyadenylated RNA confirms the upregulation in aberrantly polyadenylated histone genes in the ΔNLS-TDP-43-expressing mice. (E), Similar RT-qPCR validation with random hexamer priming shows that non-polyadenylated canonical histones are not significantly upregulated in the bigenic mice. (F), Comparison of RNA-seq and RT-PCR analysis validates RNA-seq results, except for histone transcripts (white filled symbols) which revealed distinct results when priming with random hexamers versus oligo-dT’s. *p

Techniques Used: Mouse Assay, Variant Assay, Transgenic Assay, Cross-linking Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Selection, Expressing, Random Hexamer Labeling, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

36) Product Images from "SMASH, a fragmentation and sequencing method for genomic copy number analysis"

Article Title: SMASH, a fragmentation and sequencing method for genomic copy number analysis

Journal: Genome Research

doi: 10.1101/gr.201491.115

Schematic of the SMASH method and size analysis. ( A ) Three representative genomic DNA molecules, shown in different shades of green, originate from different chromosomes or distant regions of the same chromosome. ( B ) By sonication and restriction enzyme cleavage, these molecules are fragmented into short double-stranded DNA fragments with average length of 40–50 bp, as shown in the Bioanalyzer result at right . ( C ) These short DNA pieces are then partially end-repaired and combined into longer stretches of DNA with lengths ranging from 50 bp to 7 kb. Consequently, each resulting chimeric DNA molecule contains short DNA fragments from different locations (shown by varying colors). ( D ) These DNA stretches are ligated to sequencing adaptors containing sample barcodes, shown in blue and red lines, with the open box designating the sample barcodes. ( E ) Size selection is carried out to enrich for DNA fragments in the size range of 250–700 bp, which is confirmed via Bioanalyzer. After final PCR, libraries are ready for sequencing. “FU” in the Bioanalyzer plots refers to relative fluorescence units.
Figure Legend Snippet: Schematic of the SMASH method and size analysis. ( A ) Three representative genomic DNA molecules, shown in different shades of green, originate from different chromosomes or distant regions of the same chromosome. ( B ) By sonication and restriction enzyme cleavage, these molecules are fragmented into short double-stranded DNA fragments with average length of 40–50 bp, as shown in the Bioanalyzer result at right . ( C ) These short DNA pieces are then partially end-repaired and combined into longer stretches of DNA with lengths ranging from 50 bp to 7 kb. Consequently, each resulting chimeric DNA molecule contains short DNA fragments from different locations (shown by varying colors). ( D ) These DNA stretches are ligated to sequencing adaptors containing sample barcodes, shown in blue and red lines, with the open box designating the sample barcodes. ( E ) Size selection is carried out to enrich for DNA fragments in the size range of 250–700 bp, which is confirmed via Bioanalyzer. After final PCR, libraries are ready for sequencing. “FU” in the Bioanalyzer plots refers to relative fluorescence units.

Techniques Used: Sonication, Sequencing, Selection, Polymerase Chain Reaction, Fluorescence

37) Product Images from "Identification of Genes Involved in Low Temperature-Induced Senescence of Panicle Leaf in Litchi chinensis"

Article Title: Identification of Genes Involved in Low Temperature-Induced Senescence of Panicle Leaf in Litchi chinensis

Journal: Genes

doi: 10.3390/genes10020111

Coefficient analysis of fold changes data between qRT-PCR and RNA-Seq. Scatter plots represent the fold-changes in the gene expression levels of the senescing panicle leaves compared to those of the developing panicle leaves. All the data are from the qRT-PCR analysis and RNA-Seq of the DLE, DLL, SLE, and SLL. Asterisk indicates that the slope is significantly different from zero at 0.05 probability level.
Figure Legend Snippet: Coefficient analysis of fold changes data between qRT-PCR and RNA-Seq. Scatter plots represent the fold-changes in the gene expression levels of the senescing panicle leaves compared to those of the developing panicle leaves. All the data are from the qRT-PCR analysis and RNA-Seq of the DLE, DLL, SLE, and SLL. Asterisk indicates that the slope is significantly different from zero at 0.05 probability level.

Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay, Expressing

38) Product Images from "Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma"

Article Title: Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma

Journal: Acta Neuropathologica Communications

doi: 10.1186/s40478-020-00917-6

Concordance between the results of the dPCR assay, FISH, next-generation sequencing (NGS) and LD-RT-PCR for the detection of the EGFR amplification and EGFRvIII variant in the exploratory cohort ( n = 19). a Heatmap of EGFR copy number estimated by NGS and the three dPCR assays. Each column represents a tumor sample (n = 19). The blue gradient represents the estimated value of the EGFR copy number. There is a strong agreement between the EGFR1 and EGFR2 dPCR assays and NGS. The absence of results using the NGS experiment is indicated by the light gray color. Below the heatmap, the presence of EGFR mutations and EGFRvIII variant as well as the results of FISH are presented. The presence of somatic mutations was detected by the EGFR -targeted NGS panel, and the presence of the EGFRvIII variant was detected by LD-RT-PCR. Patient #08 harbors both the EGFR amplification and EGFRvIII variant with tumor heterogeneity regarding the copy number estimation by dPCR (EGFR1 63, EGFR2 70 and EGFR3 91). b Correlation matrix plot of EGFR copy number estimation using three dPCR assays (EGFR1, EGFR2 and EGFR3) and NGS ( n = 16). The dPCR EGFR3 assay results have the highest correlation with the NGS results. On the other hand, the dPCR EGFR2 assay results have the poorest correlation, mainly due to its underestimation of the EGFR CNV in the case of EGFRvIII -positive glioblastoma. c Linear regression curves representing EGFR copy number values estimated by NGS (x-axis) and the copy number estimated by the three dPCR assays (y-axis). As expected with the results of the matrix correlation plot, the estimation using the dPCR EGFR3 assay was confirmed to have the best correlation to the NGS estimation
Figure Legend Snippet: Concordance between the results of the dPCR assay, FISH, next-generation sequencing (NGS) and LD-RT-PCR for the detection of the EGFR amplification and EGFRvIII variant in the exploratory cohort ( n = 19). a Heatmap of EGFR copy number estimated by NGS and the three dPCR assays. Each column represents a tumor sample (n = 19). The blue gradient represents the estimated value of the EGFR copy number. There is a strong agreement between the EGFR1 and EGFR2 dPCR assays and NGS. The absence of results using the NGS experiment is indicated by the light gray color. Below the heatmap, the presence of EGFR mutations and EGFRvIII variant as well as the results of FISH are presented. The presence of somatic mutations was detected by the EGFR -targeted NGS panel, and the presence of the EGFRvIII variant was detected by LD-RT-PCR. Patient #08 harbors both the EGFR amplification and EGFRvIII variant with tumor heterogeneity regarding the copy number estimation by dPCR (EGFR1 63, EGFR2 70 and EGFR3 91). b Correlation matrix plot of EGFR copy number estimation using three dPCR assays (EGFR1, EGFR2 and EGFR3) and NGS ( n = 16). The dPCR EGFR3 assay results have the highest correlation with the NGS results. On the other hand, the dPCR EGFR2 assay results have the poorest correlation, mainly due to its underestimation of the EGFR CNV in the case of EGFRvIII -positive glioblastoma. c Linear regression curves representing EGFR copy number values estimated by NGS (x-axis) and the copy number estimated by the three dPCR assays (y-axis). As expected with the results of the matrix correlation plot, the estimation using the dPCR EGFR3 assay was confirmed to have the best correlation to the NGS estimation

Techniques Used: Digital PCR, Fluorescence In Situ Hybridization, Next-Generation Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Variant Assay

39) Product Images from "Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing"

Article Title: Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr598

Targeted bisulfite sequencing. ( a ) Illustrated are the steps involved in the preparation of biotinylated RNA capture probes (top left and right), whole-genome fragment input library (top middle) and hybrid selection-enriched output library (middle left and right). The captured DNA was treated with sodium bisulfite, amplified by PCR and sequenced using an Illumina GAIIx sequencer. ( b ) Examples of targeted bisulfite sequencing using solution hybrid selection. The tracks shown from the top to bottom are: sequences of the capture probes; the DNA methylation level at each CpG site derived from the bisulfite-sequencing reads; the sequencing depth at each CpG site; RefSeq genes; and annotated CGIs in the UCSC genome browser. Red and green colors indicate methylated and unmethylated-CpG sites, respectively. MCF7-1 and MCF7-2 are technical replicates. For each sample, two tracks (methylation level and read depth) are shown. ( c ). Distribution of normalized abundance for the captured targets shared among all samples. The x -axis is the normalized abundance of each captured target, which is calculated by dividing the counts of the target by the average counts of all targets. The y -axis is the fraction of probes with coverage equal to or greater than the normalized coverage.
Figure Legend Snippet: Targeted bisulfite sequencing. ( a ) Illustrated are the steps involved in the preparation of biotinylated RNA capture probes (top left and right), whole-genome fragment input library (top middle) and hybrid selection-enriched output library (middle left and right). The captured DNA was treated with sodium bisulfite, amplified by PCR and sequenced using an Illumina GAIIx sequencer. ( b ) Examples of targeted bisulfite sequencing using solution hybrid selection. The tracks shown from the top to bottom are: sequences of the capture probes; the DNA methylation level at each CpG site derived from the bisulfite-sequencing reads; the sequencing depth at each CpG site; RefSeq genes; and annotated CGIs in the UCSC genome browser. Red and green colors indicate methylated and unmethylated-CpG sites, respectively. MCF7-1 and MCF7-2 are technical replicates. For each sample, two tracks (methylation level and read depth) are shown. ( c ). Distribution of normalized abundance for the captured targets shared among all samples. The x -axis is the normalized abundance of each captured target, which is calculated by dividing the counts of the target by the average counts of all targets. The y -axis is the fraction of probes with coverage equal to or greater than the normalized coverage.

Techniques Used: Methylation Sequencing, Selection, Amplification, Polymerase Chain Reaction, DNA Methylation Assay, Derivative Assay, Sequencing, Methylation

40) Product Images from "Liquid biopsy monitoring uncovers acquired RAS-mediated resistance to cetuximab in a substantial proportion of patients with head and neck squamous cell carcinoma"

Article Title: Liquid biopsy monitoring uncovers acquired RAS-mediated resistance to cetuximab in a substantial proportion of patients with head and neck squamous cell carcinoma

Journal: Oncotarget

doi: 10.18632/oncotarget.8943

PCR amplification of EGFR and RAS exons for Illumina targeted next generation sequencing EGFR exon 12, KRAS/NRAS exons 2/3/4 and HRAS exons 2/3 (green) were amplified from tumor tissue of 46 patients, post-cetuximab circulating tumor DNA of 20 patients and from 12 squamous carcinoma cell lines. Illumina-specific sequences for hybridization and sequencing (yellow) as well as patient-specific barcodes (red) were attached in a second PCR step.
Figure Legend Snippet: PCR amplification of EGFR and RAS exons for Illumina targeted next generation sequencing EGFR exon 12, KRAS/NRAS exons 2/3/4 and HRAS exons 2/3 (green) were amplified from tumor tissue of 46 patients, post-cetuximab circulating tumor DNA of 20 patients and from 12 squamous carcinoma cell lines. Illumina-specific sequences for hybridization and sequencing (yellow) as well as patient-specific barcodes (red) were attached in a second PCR step.

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

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Flow Cytometry:

Article Title: Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo
Article Snippet: .. During PCR amplification of the barcodes with primers that contain both Illumina adapter sequences, 4 bp indexing tags are added to allow for pooling of multiple samples per flow cell lane. .. The resultant 250 bp fragment (Figure b) contains the indexing tag, 8 bp of anchor sequence, and the 20 bp barcode, flanked by the adapters.

Amplification:

Article Title: RESA identifies mRNA regulatory sequences with high resolution
Article Snippet: .. To generate the RESA library, we isolated 3′UTR sequences by PCR amplification, induced random fragmentation by sonication, ligated Illumina sequencing adaptors, and using overlap-extension PCR introduced them into the 3′UTR of a GFP open reading frame with SP6 promoter and SV40 polyadenylation signal. ..

Article Title: Transcriptomic and GC-MS Metabolomic Analyses Reveal the Sink Strength Changes during Petunia Anther Development
Article Snippet: .. After the purification and fragmentation of RNA, cDNA synthesis, end repair, adapter ligation and PCR amplification, the cDNA library products (ranging from 200–700 bp) were sequenced on the Illumina HiSeqTM 2000 instrument using paired-end sequencing technology by the staff at Beijing Genome Institute (BGI) (Shenzhen, China). .. The datasets involving three different anther stages were deposited in the NCBI Sequence Read Archive (SRA) with Accession Number SRP126902 under BioProject Number PRJNA422657.

Article Title: Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing
Article Snippet: .. Bisulfite-modified genomic DNA from MDA-MB-231 and MCF7 was used as the template for PCR amplification of the 46 genes. .. The individual PCR products were pooled together, purified, end-repaired and ligated to the bar-coded sequencing adaptors using 454 library construction kits and sequenced according to the manufacturer's protocols (Roche 454 Life Sciences).

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: .. The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously. .. Further analyses of small RNA expression and mapping of microRNAs, piRNA, endo-siRNA, and other small RNAs were conducted pursuant to the previous method.

Article Title: Sense Transgene-Induced Post-Transcriptional Gene Silencing in Tobacco Compromises the Splicing of Endogenous Counterpart Genes
Article Snippet: .. Deep-sequencing Analysis of Small RNA The PCR amplification of the small RNA library and the nucleotide sequence of the amplified cDNA analyzed using the Illumina Genome Analyzer (GAII) were performed by Hokkaido System Science Co. Ltd. .. The adapter sequence was trimmed from the raw short-read data, and the resulting short reads (18 to 31 nt) were mapped to the nucleotide sequence of the T-DNA region or the NtFAD3 genomic region (GenBank acc. no. AB049576).

Article Title: Recurrent and functional regulatory mutations in breast cancer
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. .. After this amplification step, the product was cleaned with solid-phase reversible immobilization (SPRI) beads and quantified using PicoGreen.

Ligation:

Article Title: Transcriptomic and GC-MS Metabolomic Analyses Reveal the Sink Strength Changes during Petunia Anther Development
Article Snippet: .. After the purification and fragmentation of RNA, cDNA synthesis, end repair, adapter ligation and PCR amplification, the cDNA library products (ranging from 200–700 bp) were sequenced on the Illumina HiSeqTM 2000 instrument using paired-end sequencing technology by the staff at Beijing Genome Institute (BGI) (Shenzhen, China). .. The datasets involving three different anther stages were deposited in the NCBI Sequence Read Archive (SRA) with Accession Number SRP126902 under BioProject Number PRJNA422657.

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: .. The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously. .. Further analyses of small RNA expression and mapping of microRNAs, piRNA, endo-siRNA, and other small RNAs were conducted pursuant to the previous method.

Article Title: Recurrent and functional regulatory mutations in breast cancer
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. .. After this amplification step, the product was cleaned with solid-phase reversible immobilization (SPRI) beads and quantified using PicoGreen.

Multiple Displacement Amplification:

Article Title: Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing
Article Snippet: .. Bisulfite-modified genomic DNA from MDA-MB-231 and MCF7 was used as the template for PCR amplification of the 46 genes. .. The individual PCR products were pooled together, purified, end-repaired and ligated to the bar-coded sequencing adaptors using 454 library construction kits and sequenced according to the manufacturer's protocols (Roche 454 Life Sciences).

Isolation:

Article Title: RESA identifies mRNA regulatory sequences with high resolution
Article Snippet: .. To generate the RESA library, we isolated 3′UTR sequences by PCR amplification, induced random fragmentation by sonication, ligated Illumina sequencing adaptors, and using overlap-extension PCR introduced them into the 3′UTR of a GFP open reading frame with SP6 promoter and SV40 polyadenylation signal. ..

Purification:

Article Title: Transcriptomic and GC-MS Metabolomic Analyses Reveal the Sink Strength Changes during Petunia Anther Development
Article Snippet: .. After the purification and fragmentation of RNA, cDNA synthesis, end repair, adapter ligation and PCR amplification, the cDNA library products (ranging from 200–700 bp) were sequenced on the Illumina HiSeqTM 2000 instrument using paired-end sequencing technology by the staff at Beijing Genome Institute (BGI) (Shenzhen, China). .. The datasets involving three different anther stages were deposited in the NCBI Sequence Read Archive (SRA) with Accession Number SRP126902 under BioProject Number PRJNA422657.

Sequencing:

Article Title: RESA identifies mRNA regulatory sequences with high resolution
Article Snippet: .. To generate the RESA library, we isolated 3′UTR sequences by PCR amplification, induced random fragmentation by sonication, ligated Illumina sequencing adaptors, and using overlap-extension PCR introduced them into the 3′UTR of a GFP open reading frame with SP6 promoter and SV40 polyadenylation signal. ..

Article Title: Transcriptomic and GC-MS Metabolomic Analyses Reveal the Sink Strength Changes during Petunia Anther Development
Article Snippet: .. After the purification and fragmentation of RNA, cDNA synthesis, end repair, adapter ligation and PCR amplification, the cDNA library products (ranging from 200–700 bp) were sequenced on the Illumina HiSeqTM 2000 instrument using paired-end sequencing technology by the staff at Beijing Genome Institute (BGI) (Shenzhen, China). .. The datasets involving three different anther stages were deposited in the NCBI Sequence Read Archive (SRA) with Accession Number SRP126902 under BioProject Number PRJNA422657.

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: .. The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously. .. Further analyses of small RNA expression and mapping of microRNAs, piRNA, endo-siRNA, and other small RNAs were conducted pursuant to the previous method.

Article Title: Sense Transgene-Induced Post-Transcriptional Gene Silencing in Tobacco Compromises the Splicing of Endogenous Counterpart Genes
Article Snippet: .. Deep-sequencing Analysis of Small RNA The PCR amplification of the small RNA library and the nucleotide sequence of the amplified cDNA analyzed using the Illumina Genome Analyzer (GAII) were performed by Hokkaido System Science Co. Ltd. .. The adapter sequence was trimmed from the raw short-read data, and the resulting short reads (18 to 31 nt) were mapped to the nucleotide sequence of the T-DNA region or the NtFAD3 genomic region (GenBank acc. no. AB049576).

Article Title: Recurrent and functional regulatory mutations in breast cancer
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. .. After this amplification step, the product was cleaned with solid-phase reversible immobilization (SPRI) beads and quantified using PicoGreen.

Polymerase Chain Reaction:

Article Title: RESA identifies mRNA regulatory sequences with high resolution
Article Snippet: .. To generate the RESA library, we isolated 3′UTR sequences by PCR amplification, induced random fragmentation by sonication, ligated Illumina sequencing adaptors, and using overlap-extension PCR introduced them into the 3′UTR of a GFP open reading frame with SP6 promoter and SV40 polyadenylation signal. ..

Article Title: Transcriptomic and GC-MS Metabolomic Analyses Reveal the Sink Strength Changes during Petunia Anther Development
Article Snippet: .. After the purification and fragmentation of RNA, cDNA synthesis, end repair, adapter ligation and PCR amplification, the cDNA library products (ranging from 200–700 bp) were sequenced on the Illumina HiSeqTM 2000 instrument using paired-end sequencing technology by the staff at Beijing Genome Institute (BGI) (Shenzhen, China). .. The datasets involving three different anther stages were deposited in the NCBI Sequence Read Archive (SRA) with Accession Number SRP126902 under BioProject Number PRJNA422657.

Article Title: Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain
Article Snippet: .. An important application of gDNA-based AEI measurements is the quantification of allele-specific bias that occurs during the PCR-amplification of gDNA and cDNA segments containing the marker SNP. .. As shown in Additional file , Figure S5, significant allele-specific amplification bias for gDNA templates was observed for most genes, with occasional genes showing greater than 1.5-fold differences in amplification between alleles.

Article Title: Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing
Article Snippet: .. Bisulfite-modified genomic DNA from MDA-MB-231 and MCF7 was used as the template for PCR amplification of the 46 genes. .. The individual PCR products were pooled together, purified, end-repaired and ligated to the bar-coded sequencing adaptors using 454 library construction kits and sequenced according to the manufacturer's protocols (Roche 454 Life Sciences).

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: .. The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously. .. Further analyses of small RNA expression and mapping of microRNAs, piRNA, endo-siRNA, and other small RNAs were conducted pursuant to the previous method.

Article Title: Sense Transgene-Induced Post-Transcriptional Gene Silencing in Tobacco Compromises the Splicing of Endogenous Counterpart Genes
Article Snippet: .. Deep-sequencing Analysis of Small RNA The PCR amplification of the small RNA library and the nucleotide sequence of the amplified cDNA analyzed using the Illumina Genome Analyzer (GAII) were performed by Hokkaido System Science Co. Ltd. .. The adapter sequence was trimmed from the raw short-read data, and the resulting short reads (18 to 31 nt) were mapped to the nucleotide sequence of the T-DNA region or the NtFAD3 genomic region (GenBank acc. no. AB049576).

Article Title: Recurrent and functional regulatory mutations in breast cancer
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. .. After this amplification step, the product was cleaned with solid-phase reversible immobilization (SPRI) beads and quantified using PicoGreen.

cDNA Library Assay:

Article Title: Transcriptomic and GC-MS Metabolomic Analyses Reveal the Sink Strength Changes during Petunia Anther Development
Article Snippet: .. After the purification and fragmentation of RNA, cDNA synthesis, end repair, adapter ligation and PCR amplification, the cDNA library products (ranging from 200–700 bp) were sequenced on the Illumina HiSeqTM 2000 instrument using paired-end sequencing technology by the staff at Beijing Genome Institute (BGI) (Shenzhen, China). .. The datasets involving three different anther stages were deposited in the NCBI Sequence Read Archive (SRA) with Accession Number SRP126902 under BioProject Number PRJNA422657.

Marker:

Article Title: Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain
Article Snippet: .. An important application of gDNA-based AEI measurements is the quantification of allele-specific bias that occurs during the PCR-amplification of gDNA and cDNA segments containing the marker SNP. .. As shown in Additional file , Figure S5, significant allele-specific amplification bias for gDNA templates was observed for most genes, with occasional genes showing greater than 1.5-fold differences in amplification between alleles.

Sonication:

Article Title: RESA identifies mRNA regulatory sequences with high resolution
Article Snippet: .. To generate the RESA library, we isolated 3′UTR sequences by PCR amplification, induced random fragmentation by sonication, ligated Illumina sequencing adaptors, and using overlap-extension PCR introduced them into the 3′UTR of a GFP open reading frame with SP6 promoter and SV40 polyadenylation signal. ..

Gel Purification:

Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients
Article Snippet: .. The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously. .. Further analyses of small RNA expression and mapping of microRNAs, piRNA, endo-siRNA, and other small RNAs were conducted pursuant to the previous method.

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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 789 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr amplification/product/Illumina Inc
    Average 94 stars, based on 789 article reviews
    Price from $9.99 to $1999.99
    pcr amplification - by Bioz Stars, 2020-09
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    92
    Illumina Inc pcr amplifications
    Schematic pipelines of conventional and <t>PCR-independent</t> NGS <t>biodiversity</t> analyses.
    Pcr Amplifications, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr amplifications/product/Illumina Inc
    Average 92 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    pcr amplifications - by Bioz Stars, 2020-09
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    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

    Morphological and Phenotypic Characterization of Freshly Isolated Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids (A–C) Phase-contrast microscope revealed the morphology of the freshly isolated human pachytene spermatocytes (A), spermatogonia (B), and round spermatids (C) of OA patients. (D–F) DIC microscope showed the morphological characteristics of the freshly isolated human pachytene spermatocytes (D), spermatogonia (E), and round spermatids (F) of OA patients. Scale bars, 20 μm (A–C) and 5 μm (D–F). (G) RT-PCR revealed the transcripts of GPR125 , RET , GFRA1 , THY1 , UCHL1 , MAGEA4 , and PLZF in the fleshly isolated spermatogonia, the expression of SYCP3 and SYCP1 in pachytene spermatocytes, and mRNA of TNP1 , TNP2 , PRM1 , PRM2 , and ACR in round spermatids. RNA without RT (RT-) but with PCR of GAPDH primers was utilized as negative controls, and GAPDH served as loading controls of total RNA.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients

    doi: 10.1016/j.omtn.2017.09.007

    Figure Lengend Snippet: Morphological and Phenotypic Characterization of Freshly Isolated Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids (A–C) Phase-contrast microscope revealed the morphology of the freshly isolated human pachytene spermatocytes (A), spermatogonia (B), and round spermatids (C) of OA patients. (D–F) DIC microscope showed the morphological characteristics of the freshly isolated human pachytene spermatocytes (D), spermatogonia (E), and round spermatids (F) of OA patients. Scale bars, 20 μm (A–C) and 5 μm (D–F). (G) RT-PCR revealed the transcripts of GPR125 , RET , GFRA1 , THY1 , UCHL1 , MAGEA4 , and PLZF in the fleshly isolated spermatogonia, the expression of SYCP3 and SYCP1 in pachytene spermatocytes, and mRNA of TNP1 , TNP2 , PRM1 , PRM2 , and ACR in round spermatids. RNA without RT (RT-) but with PCR of GAPDH primers was utilized as negative controls, and GAPDH served as loading controls of total RNA.

    Article Snippet: The cDNA libraries of small RNAs were established using 100 ng of total RNA, and adaptor ligation, first-strand cDNA synthesis, PCR amplification, gel purification, and sequencing of small RNA libraries using an Illumina HiSeq 2000 were performed according to the procedure as described previously.

    Techniques: Isolation, Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction

    Schematic pipelines of conventional and PCR-independent NGS biodiversity analyses.

    Journal: GigaScience

    Article Title: Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification

    doi: 10.1186/2047-217X-2-4

    Figure Lengend Snippet: Schematic pipelines of conventional and PCR-independent NGS biodiversity analyses.

    Article Snippet: The main question of the present work is: can PCR amplifications be avoided in analyzing arthropod biodiversity using the Illumina HiSeq 2000 platform?

    Techniques: Polymerase Chain Reaction, Next-Generation Sequencing