Structured Review

Illumina Inc gdna
Exemplary workflow of three principal methodologies for TCR library preparation. The figure depicts a simplified workflow of the library preparation procedure using multiplex PCR, targeted in-solution enrichment and 5’RACE-switch-oligo nested PCR. Multiplex PCR is suitable for both <t>RNA</t> and <t>gDNA</t> sequencing. Samples undergo cDNA synthesis and 1 or more PCR steps followed by adaptor ligation and sequencing. While the forward primers for cDNA synthesis are designed to cover all known V genes for both starting materials, the location and number of the reverse primers differs, due to introns in DNA. Target enrichment, also applicable to both gDNA and RNA, is preceded by a standard library preparation including fragmentation for gDNA or mRNA purification for RNA, followed by end-repairing, A-tailing and finally adaptor ligation. The enrichment of target sequences is then performed using RNA baits complementary to the sequence of interest. The RNA baits hybridize with molecules in the library, which are then retrieved using magnetic beads and can undergo further amplification before sequencing. Nested PCR based on the 5’RACE and switch-oligo approach (only for RNA) makes use of the incorporation of an adaptor molecule at the 5′ end of the cDNA during cDNA synthesis. The forward primer for a subsequent PCR is designed to bind to the 5′ adaptor sequence, while the reverse primer is designed to bind to the C-region of the transcript. Hence, only one primer pair is required to cover the complete spectrum of possible V genes. Subsequent nested PCRs performed in the same fashion may increase outcome specificity. Finally, adaptor ligation is performed. The procedures showed in this picture constitute only an example of the different available methods
Gdna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 537 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gdna/product/Illumina Inc
Average 94 stars, based on 537 article reviews
Price from $9.99 to $1999.99
gdna - by Bioz Stars, 2020-09
94/100 stars

Images

1) Product Images from "Overview of methodologies for T-cell receptor repertoire analysis"

Article Title: Overview of methodologies for T-cell receptor repertoire analysis

Journal: BMC Biotechnology

doi: 10.1186/s12896-017-0379-9

Exemplary workflow of three principal methodologies for TCR library preparation. The figure depicts a simplified workflow of the library preparation procedure using multiplex PCR, targeted in-solution enrichment and 5’RACE-switch-oligo nested PCR. Multiplex PCR is suitable for both RNA and gDNA sequencing. Samples undergo cDNA synthesis and 1 or more PCR steps followed by adaptor ligation and sequencing. While the forward primers for cDNA synthesis are designed to cover all known V genes for both starting materials, the location and number of the reverse primers differs, due to introns in DNA. Target enrichment, also applicable to both gDNA and RNA, is preceded by a standard library preparation including fragmentation for gDNA or mRNA purification for RNA, followed by end-repairing, A-tailing and finally adaptor ligation. The enrichment of target sequences is then performed using RNA baits complementary to the sequence of interest. The RNA baits hybridize with molecules in the library, which are then retrieved using magnetic beads and can undergo further amplification before sequencing. Nested PCR based on the 5’RACE and switch-oligo approach (only for RNA) makes use of the incorporation of an adaptor molecule at the 5′ end of the cDNA during cDNA synthesis. The forward primer for a subsequent PCR is designed to bind to the 5′ adaptor sequence, while the reverse primer is designed to bind to the C-region of the transcript. Hence, only one primer pair is required to cover the complete spectrum of possible V genes. Subsequent nested PCRs performed in the same fashion may increase outcome specificity. Finally, adaptor ligation is performed. The procedures showed in this picture constitute only an example of the different available methods
Figure Legend Snippet: Exemplary workflow of three principal methodologies for TCR library preparation. The figure depicts a simplified workflow of the library preparation procedure using multiplex PCR, targeted in-solution enrichment and 5’RACE-switch-oligo nested PCR. Multiplex PCR is suitable for both RNA and gDNA sequencing. Samples undergo cDNA synthesis and 1 or more PCR steps followed by adaptor ligation and sequencing. While the forward primers for cDNA synthesis are designed to cover all known V genes for both starting materials, the location and number of the reverse primers differs, due to introns in DNA. Target enrichment, also applicable to both gDNA and RNA, is preceded by a standard library preparation including fragmentation for gDNA or mRNA purification for RNA, followed by end-repairing, A-tailing and finally adaptor ligation. The enrichment of target sequences is then performed using RNA baits complementary to the sequence of interest. The RNA baits hybridize with molecules in the library, which are then retrieved using magnetic beads and can undergo further amplification before sequencing. Nested PCR based on the 5’RACE and switch-oligo approach (only for RNA) makes use of the incorporation of an adaptor molecule at the 5′ end of the cDNA during cDNA synthesis. The forward primer for a subsequent PCR is designed to bind to the 5′ adaptor sequence, while the reverse primer is designed to bind to the C-region of the transcript. Hence, only one primer pair is required to cover the complete spectrum of possible V genes. Subsequent nested PCRs performed in the same fashion may increase outcome specificity. Finally, adaptor ligation is performed. The procedures showed in this picture constitute only an example of the different available methods

Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Nested PCR, Sequencing, Ligation, Purification, Magnetic Beads, Amplification

2) Product Images from "In vivo loss-of-function screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer"

Article Title: In vivo loss-of-function screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer

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

doi: 10.1073/pnas.1705441114

An in vivo loss-of-function screen identifies potent drug targets for EOC. ( A ) Diagram of the in vivo pooled library screen. A large-scale pooled shRNA library targeting 7,490 druggable genes with 42,450 shRNAs was transduced into SKOV3 cells at an MOI of 0.2. Five million transduced cells then were injected into 12 female nude mice i.p., and peritoneal tumors were harvested 7 wk after injection. Genomic DNA extracted from referential transduced cells, and the 12 biggest tumors, were amplified and sequenced. ( B ) The average numbers of detected shRNAs from the first and second screen are shown. ( C ) The top 10 genes, which had multiple shRNAs showing substantial depletion in PC tumors, are ranked according to the average FC of shRNAs read counts in tumors relative to cells.
Figure Legend Snippet: An in vivo loss-of-function screen identifies potent drug targets for EOC. ( A ) Diagram of the in vivo pooled library screen. A large-scale pooled shRNA library targeting 7,490 druggable genes with 42,450 shRNAs was transduced into SKOV3 cells at an MOI of 0.2. Five million transduced cells then were injected into 12 female nude mice i.p., and peritoneal tumors were harvested 7 wk after injection. Genomic DNA extracted from referential transduced cells, and the 12 biggest tumors, were amplified and sequenced. ( B ) The average numbers of detected shRNAs from the first and second screen are shown. ( C ) The top 10 genes, which had multiple shRNAs showing substantial depletion in PC tumors, are ranked according to the average FC of shRNAs read counts in tumors relative to cells.

Techniques Used: In Vivo, shRNA, Injection, Mouse Assay, Amplification

3) Product Images from "Direct Targets of CodY in Staphylococcus aureus ▿ ▿ †"

Article Title: Direct Targets of CodY in Staphylococcus aureus ▿ ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.00220-10

Protocol for genome-wide identification of CodY binding sites. Briefly, gDNA was sheared by sonication. Adapters were ligated to the sheared DNA and then gel purified to obtain a DNA library containing fragments of 400 to 500 bp in length. The DNA library was then subjected to PCR amplification using adapter-specific primers to generate enough DNA for the pull-down experiment. A 200 nM concentration of His-tagged CodY or 200 nM His-tagged Acn was incubated with 50 μg sheared, adapter-ligated DNA and the effector molecules (2 mM GTP and 10 mM ILV). Protein-DNA complexes were purified with a Co 2+ resin. Following elution of the protein from the resin, the DNA was isolated, PCR amplified again, and subjected to analysis with a Illumina Genome Analyzer II.
Figure Legend Snippet: Protocol for genome-wide identification of CodY binding sites. Briefly, gDNA was sheared by sonication. Adapters were ligated to the sheared DNA and then gel purified to obtain a DNA library containing fragments of 400 to 500 bp in length. The DNA library was then subjected to PCR amplification using adapter-specific primers to generate enough DNA for the pull-down experiment. A 200 nM concentration of His-tagged CodY or 200 nM His-tagged Acn was incubated with 50 μg sheared, adapter-ligated DNA and the effector molecules (2 mM GTP and 10 mM ILV). Protein-DNA complexes were purified with a Co 2+ resin. Following elution of the protein from the resin, the DNA was isolated, PCR amplified again, and subjected to analysis with a Illumina Genome Analyzer II.

Techniques Used: Genome Wide, Binding Assay, Sonication, Purification, Polymerase Chain Reaction, Amplification, Concentration Assay, Incubation, Isolation

4) Product Images from "Characterisation of the changing genomic landscape of metastatic melanoma using cell free DNA"

Article Title: Characterisation of the changing genomic landscape of metastatic melanoma using cell free DNA

Journal: NPJ genomic medicine

doi: 10.1038/s41525-017-0030-7

Coverage uniformity of WGS libraries. a Represented is the cumulative proportion of sequencing coverage per cumulative proportion of sequence in whole genome sequencing across normal germline DNA (gDNA) from peripheral blood mononuclear cells, two cfDNA
Figure Legend Snippet: Coverage uniformity of WGS libraries. a Represented is the cumulative proportion of sequencing coverage per cumulative proportion of sequence in whole genome sequencing across normal germline DNA (gDNA) from peripheral blood mononuclear cells, two cfDNA

Techniques Used: Sequencing

5) Product Images from "High Quality Genome-Wide Genotyping from Archived Dried Blood Spots without DNA Amplification"

Article Title: High Quality Genome-Wide Genotyping from Archived Dried Blood Spots without DNA Amplification

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064710

Genotyping performance as a function of gDNA concentration. The genotyping performance of the gDNA samples (A) and wgaDNA samples (B) described in Figure 1 is plotted against the DNA concentration of the sample as determined by Qubit for gDNA and Picogreen for wgaDNA (bottom axis) and total amount of DNA used (4 µl) during each genotyping run (top axis). Picogreen was used for wgaDNA because Qubit does not discern concentrations above 100 ng/µl. Note that all gDNA samples with DNA concentrations above 5 ng/µl (∼20 ng DNA) were genotyped successfully (A). No similar threshold was observed for wgaDNA samples (B).
Figure Legend Snippet: Genotyping performance as a function of gDNA concentration. The genotyping performance of the gDNA samples (A) and wgaDNA samples (B) described in Figure 1 is plotted against the DNA concentration of the sample as determined by Qubit for gDNA and Picogreen for wgaDNA (bottom axis) and total amount of DNA used (4 µl) during each genotyping run (top axis). Picogreen was used for wgaDNA because Qubit does not discern concentrations above 100 ng/µl. Note that all gDNA samples with DNA concentrations above 5 ng/µl (∼20 ng DNA) were genotyped successfully (A). No similar threshold was observed for wgaDNA samples (B).

Techniques Used: Concentration Assay

Success and accuracy of genotyping with genomic or amplified DNA from dried blood spots. Genomic DNA (gDNA) extracted from two 2 mm punches obtained from archived dried blood spots (DBS) from nine individuals (1–9) was genotyped on an Illumina Infinium 300,000 SNP test chip (set “a”, 1a–9a). For six of the DBS (1,3,4,5,7,8), a set (set “b”) of extraction duplicates (duplicate extractions from the same DBS) was prepared and genotyped in the same way. A 1 µl aliquot of each of these 15 samples of gDNA was whole genome amplified (wgaDNA) and then genotyped in the same way as sets “a” and “b”: set “c” wgaDNA samples (1c–9c) were amplified from set “a” gDNA samples, and set “d” wgaDNA samples (1d, 3d, 4d, 5d, 7d, 8d) were amplified from set “b” gDNA samples. (A) Performance quality of the gDNA and wgaDNA samples on genome-wide SNP genotyping. Successful, Marginal, and Failed sample performance parameters are given in Results. (B) SNP genotyping replication error. The number of SNP discrepancies for each sample was determined using gDNA set “a” as the reference. Replication error (the number of discrepancies among SNPs called in both the sample and reference divided by the total number of SNPs called in both) was 1.6×10 −5 for set “b”, 1.5×10 −4 for set “c”, and 1.2×10 −4 for set “d”. These replication error values are provisional because we do not know the true genotype of the discrepant loci and there may be gDNA-specific and/or wgaDNA-specific artifacts.
Figure Legend Snippet: Success and accuracy of genotyping with genomic or amplified DNA from dried blood spots. Genomic DNA (gDNA) extracted from two 2 mm punches obtained from archived dried blood spots (DBS) from nine individuals (1–9) was genotyped on an Illumina Infinium 300,000 SNP test chip (set “a”, 1a–9a). For six of the DBS (1,3,4,5,7,8), a set (set “b”) of extraction duplicates (duplicate extractions from the same DBS) was prepared and genotyped in the same way. A 1 µl aliquot of each of these 15 samples of gDNA was whole genome amplified (wgaDNA) and then genotyped in the same way as sets “a” and “b”: set “c” wgaDNA samples (1c–9c) were amplified from set “a” gDNA samples, and set “d” wgaDNA samples (1d, 3d, 4d, 5d, 7d, 8d) were amplified from set “b” gDNA samples. (A) Performance quality of the gDNA and wgaDNA samples on genome-wide SNP genotyping. Successful, Marginal, and Failed sample performance parameters are given in Results. (B) SNP genotyping replication error. The number of SNP discrepancies for each sample was determined using gDNA set “a” as the reference. Replication error (the number of discrepancies among SNPs called in both the sample and reference divided by the total number of SNPs called in both) was 1.6×10 −5 for set “b”, 1.5×10 −4 for set “c”, and 1.2×10 −4 for set “d”. These replication error values are provisional because we do not know the true genotype of the discrepant loci and there may be gDNA-specific and/or wgaDNA-specific artifacts.

Techniques Used: Amplification, Chromatin Immunoprecipitation, Genome Wide

6) Product Images from "Dietary Flavanols Modulate the Transcription of Genes Associated with Cardiovascular Pathology without Changes in Their DNA Methylation State"

Article Title: Dietary Flavanols Modulate the Transcription of Genes Associated with Cardiovascular Pathology without Changes in Their DNA Methylation State

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095527

CpG pyrosequencing-based validation of Illumina 450K array results. DNA methylation of blood leukocyte gDNA of 5 volunteers was bisulfite treated and amplified by specific biotinylated primer sets for respectively a highly methylated CpG probe region of the CCR6 gene, a medium methylated CpG probe region of the PCDHB4 gene and a weakly methylated CpG probe region of the MAFB gene. DNA methylation intensities obtained from the different CpG probes by CpG pyrosequencing were plotted against the β-values (0
Figure Legend Snippet: CpG pyrosequencing-based validation of Illumina 450K array results. DNA methylation of blood leukocyte gDNA of 5 volunteers was bisulfite treated and amplified by specific biotinylated primer sets for respectively a highly methylated CpG probe region of the CCR6 gene, a medium methylated CpG probe region of the PCDHB4 gene and a weakly methylated CpG probe region of the MAFB gene. DNA methylation intensities obtained from the different CpG probes by CpG pyrosequencing were plotted against the β-values (0

Techniques Used: DNA Methylation Assay, Amplification, Methylation

Intra-individual DNA methylation before and after MOF supplementation. Genome wide DNA methylation profiles of blood leukocyte gDNA of 10 individuals were profiled by Illumina 450K CpG array. A) DNA methylation intensities (β-values 0
Figure Legend Snippet: Intra-individual DNA methylation before and after MOF supplementation. Genome wide DNA methylation profiles of blood leukocyte gDNA of 10 individuals were profiled by Illumina 450K CpG array. A) DNA methylation intensities (β-values 0

Techniques Used: DNA Methylation Assay, Genome Wide

7) Product Images from "Extreme MHC class I diversity in the sedge warbler (Acrocephalus schoenobaenus); selection patterns and allelic divergence suggest that different genes have different functions"

Article Title: Extreme MHC class I diversity in the sedge warbler (Acrocephalus schoenobaenus); selection patterns and allelic divergence suggest that different genes have different functions

Journal: BMC Evolutionary Biology

doi: 10.1186/s12862-017-0997-9

Neighbor-net network of sedge warbler MHC class I exon 3 alleles constructed from all cDNA and gDNA alleles detected in four individuals, alleles marked with bold and italics were found only in gDNA amplicons. Bootstrap support values (based on 1000 replicates) higher than 70% are presented. The loops imply areas of phylogenetic uncertainty or reticulations. 3 bp deletion alleles are shown in orange , 6 bp deletion alleles in green and alleles with no deletion in blue
Figure Legend Snippet: Neighbor-net network of sedge warbler MHC class I exon 3 alleles constructed from all cDNA and gDNA alleles detected in four individuals, alleles marked with bold and italics were found only in gDNA amplicons. Bootstrap support values (based on 1000 replicates) higher than 70% are presented. The loops imply areas of phylogenetic uncertainty or reticulations. 3 bp deletion alleles are shown in orange , 6 bp deletion alleles in green and alleles with no deletion in blue

Techniques Used: Construct

8) Product Images from "Plasma membrane associated transcription of cytoplasmic DNA"

Article Title: Plasma membrane associated transcription of cytoplasmic DNA

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

doi: 10.1073/pnas.1208716109

Deep sequencing of cmDNA. ( A ) Distribution in human chromosomes of DNA sequences of α-satellite repeat, pericentromeric, subtelomeric, and ribosomal RNA fragments that were highly enriched in cmDNA compared with gDNA. LSU-rRNA, large subunit ribosomal
Figure Legend Snippet: Deep sequencing of cmDNA. ( A ) Distribution in human chromosomes of DNA sequences of α-satellite repeat, pericentromeric, subtelomeric, and ribosomal RNA fragments that were highly enriched in cmDNA compared with gDNA. LSU-rRNA, large subunit ribosomal

Techniques Used: Sequencing

9) Product Images from "SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states"

Article Title: SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states

Journal: BMC Genomics

doi: 10.1186/s12864-018-4530-3

NGS library construction with alternatively sheared gDNA by using SALP method. a Schematic of library construction with the enzymatically digested and sonicated DNA by using SALP method. b Distribution of reads density of NGS library constructed with the Hind III-digested HepG2 gDNA and the density of predicted Hind III restriction sites in whole genome. c : Figure S7
Figure Legend Snippet: NGS library construction with alternatively sheared gDNA by using SALP method. a Schematic of library construction with the enzymatically digested and sonicated DNA by using SALP method. b Distribution of reads density of NGS library constructed with the Hind III-digested HepG2 gDNA and the density of predicted Hind III restriction sites in whole genome. c : Figure S7

Techniques Used: Next-Generation Sequencing, Sonication, Construct

10) Product Images from "OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development"

Article Title: OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development

Journal: eLife

doi: 10.7554/eLife.34870

The TET1-OGT interaction promotes TET1 function in the zebrafish embryo. ( A ) Representative images of runx1 labeling in the dorsal aorta of wild type or tet2/3 DM zebrafish embryos, uninjected or injected with mRNA encoding mouse Tet1 wild type or D2018A. ( B ) Percentage of embryos with high runx1 expression along the dorsal aorta. ( C ) 5hmC dot blot of genomic DNA from wild type or tet2/3 DM zebrafish embryos injected with Tet1 wild type or D2018A mRNA. Methylene blue was used as a loading control. ( D ) Quantification of 5hmC levels from three dot blots, normalized to methylene blue staining. Error bars denote s.d. *p
Figure Legend Snippet: The TET1-OGT interaction promotes TET1 function in the zebrafish embryo. ( A ) Representative images of runx1 labeling in the dorsal aorta of wild type or tet2/3 DM zebrafish embryos, uninjected or injected with mRNA encoding mouse Tet1 wild type or D2018A. ( B ) Percentage of embryos with high runx1 expression along the dorsal aorta. ( C ) 5hmC dot blot of genomic DNA from wild type or tet2/3 DM zebrafish embryos injected with Tet1 wild type or D2018A mRNA. Methylene blue was used as a loading control. ( D ) Quantification of 5hmC levels from three dot blots, normalized to methylene blue staining. Error bars denote s.d. *p

Techniques Used: Labeling, Injection, Expressing, Dot Blot, Staining

11) Product Images from "DNA Methylome Profiling on the Infinium HumanMethylation450 Array from Limiting Quantities of Genomic DNA from a Single, Small Archived Bloodspot"

Article Title: DNA Methylome Profiling on the Infinium HumanMethylation450 Array from Limiting Quantities of Genomic DNA from a Single, Small Archived Bloodspot

Journal: Genetic Testing and Molecular Biomarkers

doi: 10.1089/gtmb.2017.0019

Reproducibility of DNA methylation profiles from 25 ng of gDNA isolated from small archived dried bloodspots. (A) Correlation plot of array β-values from 25 ng replicate samples. (B) Correlation plot of array β-values between two independent samples representing the median correlation coefficient (0.9873) from all pair-wise comparisons ( n = 190) of 20 individuals. (C) Distribution of the standard deviations of mean β-values obtained from averaging array loci data from 20 individuals.
Figure Legend Snippet: Reproducibility of DNA methylation profiles from 25 ng of gDNA isolated from small archived dried bloodspots. (A) Correlation plot of array β-values from 25 ng replicate samples. (B) Correlation plot of array β-values between two independent samples representing the median correlation coefficient (0.9873) from all pair-wise comparisons ( n = 190) of 20 individuals. (C) Distribution of the standard deviations of mean β-values obtained from averaging array loci data from 20 individuals.

Techniques Used: DNA Methylation Assay, Isolation

12) Product Images from "High Quality Genome-Wide Genotyping from Archived Dried Blood Spots without DNA Amplification"

Article Title: High Quality Genome-Wide Genotyping from Archived Dried Blood Spots without DNA Amplification

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064710

Genotyping performance as a function of gDNA concentration. The genotyping performance of the gDNA samples (A) and wgaDNA samples (B) described in Figure 1 is plotted against the DNA concentration of the sample as determined by Qubit for gDNA and Picogreen for wgaDNA (bottom axis) and total amount of DNA used (4 µl) during each genotyping run (top axis). Picogreen was used for wgaDNA because Qubit does not discern concentrations above 100 ng/µl. Note that all gDNA samples with DNA concentrations above 5 ng/µl (∼20 ng DNA) were genotyped successfully (A). No similar threshold was observed for wgaDNA samples (B).
Figure Legend Snippet: Genotyping performance as a function of gDNA concentration. The genotyping performance of the gDNA samples (A) and wgaDNA samples (B) described in Figure 1 is plotted against the DNA concentration of the sample as determined by Qubit for gDNA and Picogreen for wgaDNA (bottom axis) and total amount of DNA used (4 µl) during each genotyping run (top axis). Picogreen was used for wgaDNA because Qubit does not discern concentrations above 100 ng/µl. Note that all gDNA samples with DNA concentrations above 5 ng/µl (∼20 ng DNA) were genotyped successfully (A). No similar threshold was observed for wgaDNA samples (B).

Techniques Used: Concentration Assay

Reproducibility of gDNA yields from the same dried blood spot. gDNA was extracted from two 2 mm punches of seven different archived DBS (1–7), and the process was repeated with fresh punches from the same DBS on a separate day. DNA was quantitated by Qubit. Different DBS showed ∼5-fold difference in DNA yield, whereas extraction duplicates of the same DBS gave almost identical DNA yield (103% ±15%, mean ± S.D.).
Figure Legend Snippet: Reproducibility of gDNA yields from the same dried blood spot. gDNA was extracted from two 2 mm punches of seven different archived DBS (1–7), and the process was repeated with fresh punches from the same DBS on a separate day. DNA was quantitated by Qubit. Different DBS showed ∼5-fold difference in DNA yield, whereas extraction duplicates of the same DBS gave almost identical DNA yield (103% ±15%, mean ± S.D.).

Techniques Used:

gDNA yield increases with increasing number of dried blood spot punches. The indicated number of punches was obtained from 19 archived DBS for gDNA extraction. DNA yield was determined by Qubit ( Table S1 ) and the values (mean ± S.D.) were normalized to the yield for a single punch. p
Figure Legend Snippet: gDNA yield increases with increasing number of dried blood spot punches. The indicated number of punches was obtained from 19 archived DBS for gDNA extraction. DNA yield was determined by Qubit ( Table S1 ) and the values (mean ± S.D.) were normalized to the yield for a single punch. p

Techniques Used:

Success and accuracy of genotyping with genomic or amplified DNA from dried blood spots. Genomic DNA (gDNA) extracted from two 2 mm punches obtained from archived dried blood spots (DBS) from nine individuals (1–9) was genotyped on an Illumina Infinium 300,000 SNP test chip (set “a”, 1a–9a). For six of the DBS (1,3,4,5,7,8), a set (set “b”) of extraction duplicates (duplicate extractions from the same DBS) was prepared and genotyped in the same way. A 1 µl aliquot of each of these 15 samples of gDNA was whole genome amplified (wgaDNA) and then genotyped in the same way as sets “a” and “b”: set “c” wgaDNA samples (1c–9c) were amplified from set “a” gDNA samples, and set “d” wgaDNA samples (1d, 3d, 4d, 5d, 7d, 8d) were amplified from set “b” gDNA samples. (A) Performance quality of the gDNA and wgaDNA samples on genome-wide SNP genotyping. Successful, Marginal, and Failed sample performance parameters are given in Results. (B) SNP genotyping replication error. The number of SNP discrepancies for each sample was determined using gDNA set “a” as the reference. Replication error (the number of discrepancies among SNPs called in both the sample and reference divided by the total number of SNPs called in both) was 1.6×10 −5 for set “b”, 1.5×10 −4 for set “c”, and 1.2×10 −4 for set “d”. These replication error values are provisional because we do not know the true genotype of the discrepant loci and there may be gDNA-specific and/or wgaDNA-specific artifacts.
Figure Legend Snippet: Success and accuracy of genotyping with genomic or amplified DNA from dried blood spots. Genomic DNA (gDNA) extracted from two 2 mm punches obtained from archived dried blood spots (DBS) from nine individuals (1–9) was genotyped on an Illumina Infinium 300,000 SNP test chip (set “a”, 1a–9a). For six of the DBS (1,3,4,5,7,8), a set (set “b”) of extraction duplicates (duplicate extractions from the same DBS) was prepared and genotyped in the same way. A 1 µl aliquot of each of these 15 samples of gDNA was whole genome amplified (wgaDNA) and then genotyped in the same way as sets “a” and “b”: set “c” wgaDNA samples (1c–9c) were amplified from set “a” gDNA samples, and set “d” wgaDNA samples (1d, 3d, 4d, 5d, 7d, 8d) were amplified from set “b” gDNA samples. (A) Performance quality of the gDNA and wgaDNA samples on genome-wide SNP genotyping. Successful, Marginal, and Failed sample performance parameters are given in Results. (B) SNP genotyping replication error. The number of SNP discrepancies for each sample was determined using gDNA set “a” as the reference. Replication error (the number of discrepancies among SNPs called in both the sample and reference divided by the total number of SNPs called in both) was 1.6×10 −5 for set “b”, 1.5×10 −4 for set “c”, and 1.2×10 −4 for set “d”. These replication error values are provisional because we do not know the true genotype of the discrepant loci and there may be gDNA-specific and/or wgaDNA-specific artifacts.

Techniques Used: Amplification, Chromatin Immunoprecipitation, Genome Wide

13) Product Images from "High Quality Genome-Wide Genotyping from Archived Dried Blood Spots without DNA Amplification"

Article Title: High Quality Genome-Wide Genotyping from Archived Dried Blood Spots without DNA Amplification

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064710

Genotyping performance as a function of gDNA concentration. The genotyping performance of the gDNA samples (A) and wgaDNA samples (B) described in Figure 1 is plotted against the DNA concentration of the sample as determined by Qubit for gDNA and Picogreen for wgaDNA (bottom axis) and total amount of DNA used (4 µl) during each genotyping run (top axis). Picogreen was used for wgaDNA because Qubit does not discern concentrations above 100 ng/µl. Note that all gDNA samples with DNA concentrations above 5 ng/µl (∼20 ng DNA) were genotyped successfully (A). No similar threshold was observed for wgaDNA samples (B).
Figure Legend Snippet: Genotyping performance as a function of gDNA concentration. The genotyping performance of the gDNA samples (A) and wgaDNA samples (B) described in Figure 1 is plotted against the DNA concentration of the sample as determined by Qubit for gDNA and Picogreen for wgaDNA (bottom axis) and total amount of DNA used (4 µl) during each genotyping run (top axis). Picogreen was used for wgaDNA because Qubit does not discern concentrations above 100 ng/µl. Note that all gDNA samples with DNA concentrations above 5 ng/µl (∼20 ng DNA) were genotyped successfully (A). No similar threshold was observed for wgaDNA samples (B).

Techniques Used: Concentration Assay

Reproducibility of gDNA yields from the same dried blood spot. gDNA was extracted from two 2 mm punches of seven different archived DBS (1–7), and the process was repeated with fresh punches from the same DBS on a separate day. DNA was quantitated by Qubit. Different DBS showed ∼5-fold difference in DNA yield, whereas extraction duplicates of the same DBS gave almost identical DNA yield (103% ±15%, mean ± S.D.).
Figure Legend Snippet: Reproducibility of gDNA yields from the same dried blood spot. gDNA was extracted from two 2 mm punches of seven different archived DBS (1–7), and the process was repeated with fresh punches from the same DBS on a separate day. DNA was quantitated by Qubit. Different DBS showed ∼5-fold difference in DNA yield, whereas extraction duplicates of the same DBS gave almost identical DNA yield (103% ±15%, mean ± S.D.).

Techniques Used:

gDNA yield increases with increasing number of dried blood spot punches. The indicated number of punches was obtained from 19 archived DBS for gDNA extraction. DNA yield was determined by Qubit ( Table S1 ) and the values (mean ± S.D.) were normalized to the yield for a single punch. p
Figure Legend Snippet: gDNA yield increases with increasing number of dried blood spot punches. The indicated number of punches was obtained from 19 archived DBS for gDNA extraction. DNA yield was determined by Qubit ( Table S1 ) and the values (mean ± S.D.) were normalized to the yield for a single punch. p

Techniques Used:

Success and accuracy of genotyping with genomic or amplified DNA from dried blood spots. Genomic DNA (gDNA) extracted from two 2 mm punches obtained from archived dried blood spots (DBS) from nine individuals (1–9) was genotyped on an Illumina Infinium 300,000 SNP test chip (set “a”, 1a–9a). For six of the DBS (1,3,4,5,7,8), a set (set “b”) of extraction duplicates (duplicate extractions from the same DBS) was prepared and genotyped in the same way. A 1 µl aliquot of each of these 15 samples of gDNA was whole genome amplified (wgaDNA) and then genotyped in the same way as sets “a” and “b”: set “c” wgaDNA samples (1c–9c) were amplified from set “a” gDNA samples, and set “d” wgaDNA samples (1d, 3d, 4d, 5d, 7d, 8d) were amplified from set “b” gDNA samples. (A) Performance quality of the gDNA and wgaDNA samples on genome-wide SNP genotyping. Successful, Marginal, and Failed sample performance parameters are given in Results. (B) SNP genotyping replication error. The number of SNP discrepancies for each sample was determined using gDNA set “a” as the reference. Replication error (the number of discrepancies among SNPs called in both the sample and reference divided by the total number of SNPs called in both) was 1.6×10 −5 for set “b”, 1.5×10 −4 for set “c”, and 1.2×10 −4 for set “d”. These replication error values are provisional because we do not know the true genotype of the discrepant loci and there may be gDNA-specific and/or wgaDNA-specific artifacts.
Figure Legend Snippet: Success and accuracy of genotyping with genomic or amplified DNA from dried blood spots. Genomic DNA (gDNA) extracted from two 2 mm punches obtained from archived dried blood spots (DBS) from nine individuals (1–9) was genotyped on an Illumina Infinium 300,000 SNP test chip (set “a”, 1a–9a). For six of the DBS (1,3,4,5,7,8), a set (set “b”) of extraction duplicates (duplicate extractions from the same DBS) was prepared and genotyped in the same way. A 1 µl aliquot of each of these 15 samples of gDNA was whole genome amplified (wgaDNA) and then genotyped in the same way as sets “a” and “b”: set “c” wgaDNA samples (1c–9c) were amplified from set “a” gDNA samples, and set “d” wgaDNA samples (1d, 3d, 4d, 5d, 7d, 8d) were amplified from set “b” gDNA samples. (A) Performance quality of the gDNA and wgaDNA samples on genome-wide SNP genotyping. Successful, Marginal, and Failed sample performance parameters are given in Results. (B) SNP genotyping replication error. The number of SNP discrepancies for each sample was determined using gDNA set “a” as the reference. Replication error (the number of discrepancies among SNPs called in both the sample and reference divided by the total number of SNPs called in both) was 1.6×10 −5 for set “b”, 1.5×10 −4 for set “c”, and 1.2×10 −4 for set “d”. These replication error values are provisional because we do not know the true genotype of the discrepant loci and there may be gDNA-specific and/or wgaDNA-specific artifacts.

Techniques Used: Amplification, Chromatin Immunoprecipitation, Genome Wide

14) Product Images from "The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics of regulatory sequences"

Article Title: The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics of regulatory sequences

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1214

Well-positioned nucleosomes primarily localize to regulatory regions and display sequence-driven rotational positioning. ( A ) Screenshot displaying coverage plots of sub + mono-, sub-, mono-, supra-, inter- and di-nucleosomal range MNase-seq fragments as well as sub + mono range H4-ChIP-seq fragments corrected by matching gDNA control. Region shown is chr6:92 500–106 500. Blue gene: forward strand; Red gene: reverse strand; Green blocks: 10 000 most positioned nucleosomes as identified by DANPOS ( B ) Pie charts displaying the distribution of gDNA (left), all nucleosomes identified by DANPOS (middle) or the 10 000 most positioned nucleosomes (right) in various genomic regions (single-exon genes, multi-exon genes, 5′ intergenic regions, 3′ intergenic regions), respectively ( C ) Dinucleotide frequency profiles in and around 147 bp gDNA fragments (left), or MNase-seq fragments overlapping all (middle) or the 10 000 most positioned (right) nucleosomes called by DANPOS. Note: this analysis was performed exclusively on 147 bp long fragments selected based on paired-end mapping. Grey vertical lines are positioned in 10.5 bp intervals starting 4 bp inwards from the fragment ends. Note: the tri-nucleotide frequency observable in the middle panel is likely caused by codon bias in nucleosomes originating from coding sequences.
Figure Legend Snippet: Well-positioned nucleosomes primarily localize to regulatory regions and display sequence-driven rotational positioning. ( A ) Screenshot displaying coverage plots of sub + mono-, sub-, mono-, supra-, inter- and di-nucleosomal range MNase-seq fragments as well as sub + mono range H4-ChIP-seq fragments corrected by matching gDNA control. Region shown is chr6:92 500–106 500. Blue gene: forward strand; Red gene: reverse strand; Green blocks: 10 000 most positioned nucleosomes as identified by DANPOS ( B ) Pie charts displaying the distribution of gDNA (left), all nucleosomes identified by DANPOS (middle) or the 10 000 most positioned nucleosomes (right) in various genomic regions (single-exon genes, multi-exon genes, 5′ intergenic regions, 3′ intergenic regions), respectively ( C ) Dinucleotide frequency profiles in and around 147 bp gDNA fragments (left), or MNase-seq fragments overlapping all (middle) or the 10 000 most positioned (right) nucleosomes called by DANPOS. Note: this analysis was performed exclusively on 147 bp long fragments selected based on paired-end mapping. Grey vertical lines are positioned in 10.5 bp intervals starting 4 bp inwards from the fragment ends. Note: the tri-nucleotide frequency observable in the middle panel is likely caused by codon bias in nucleosomes originating from coding sequences.

Techniques Used: Sequencing, Chromatin Immunoprecipitation, End-sequence Profiling

The nucleosome landscape can be predictive for functional DNA motifs. Smoothened, gDNA-corrected average nucleosome occupancy profiles around the top 394 or 726 scoring occurrences with P -value ≤ 8.43 × 10 −5 of the TGCATGCA motif that are located in ( A ) upstream or ( B ) coding regions. ( C and D ) Screenshot of the nucleosome landscape (MNase-Seq-over-gDNA ratio for sub + mono-nucleosomal fragments) around two TGCATGCA motifs. Motif +/− 25 nt upstream of genes PF3D7_1312700 (C) and PF3D7_0114000 (D) is highlighted (blue and pink) and used for DNA pulldown experiments. Blue gene: forward strand; Red gene: reverse strand. ( E and F ) Scatterplot displaying the quantitative proteomic analysis of duplicate, label-swap DNA pulldowns performed with 500 μg mixed-stage nuclear extract. Statistically significant outliers (FDR
Figure Legend Snippet: The nucleosome landscape can be predictive for functional DNA motifs. Smoothened, gDNA-corrected average nucleosome occupancy profiles around the top 394 or 726 scoring occurrences with P -value ≤ 8.43 × 10 −5 of the TGCATGCA motif that are located in ( A ) upstream or ( B ) coding regions. ( C and D ) Screenshot of the nucleosome landscape (MNase-Seq-over-gDNA ratio for sub + mono-nucleosomal fragments) around two TGCATGCA motifs. Motif +/− 25 nt upstream of genes PF3D7_1312700 (C) and PF3D7_0114000 (D) is highlighted (blue and pink) and used for DNA pulldown experiments. Blue gene: forward strand; Red gene: reverse strand. ( E and F ) Scatterplot displaying the quantitative proteomic analysis of duplicate, label-swap DNA pulldowns performed with 500 μg mixed-stage nuclear extract. Statistically significant outliers (FDR

Techniques Used: Functional Assay

Positioning of nucleosomes on landmark transcriptional sites is largely transcription-independent. ( A ) Individual (heatmap, T40, log2-MNase-Seq/gDNA ratio) and average (line-graph, T-all) MNase-seq occupancy profiles around transcriptional landmark sites normalized by gDNA. The bottom panels show corresponding GC-content profiles. Ten-thousand non-overlapping random sites were selected uniformly across the genome as control. Note: for TSS and TTS individual occupancy profiles are sorted based on the mRNA abundance of the corresponding gene, while all other profiles are sorted based on distance to another landmark site (as indicated). Dashed grey line indicates the approximate position of the neighbouring landmark site. Heatmaps displaying individual profiles for all landmark sites sorted in three different ways (hierarchical clustering, mRNA abundance, distance to nearest landmark site) can be found in Supplementary Figure S4. ( B ) Frequency profiles of longer than 6 nt homopolymeric tracks (poly dA (red), poly dT (blue)) around transcription landmark sites. ( C ) Average normalized profiles (top) and GC-content profiles (bottom) for euchromatic genes in T40 separated into four different mRNA abundance quartiles. Darker colours reflect higher mRNA abundance.
Figure Legend Snippet: Positioning of nucleosomes on landmark transcriptional sites is largely transcription-independent. ( A ) Individual (heatmap, T40, log2-MNase-Seq/gDNA ratio) and average (line-graph, T-all) MNase-seq occupancy profiles around transcriptional landmark sites normalized by gDNA. The bottom panels show corresponding GC-content profiles. Ten-thousand non-overlapping random sites were selected uniformly across the genome as control. Note: for TSS and TTS individual occupancy profiles are sorted based on the mRNA abundance of the corresponding gene, while all other profiles are sorted based on distance to another landmark site (as indicated). Dashed grey line indicates the approximate position of the neighbouring landmark site. Heatmaps displaying individual profiles for all landmark sites sorted in three different ways (hierarchical clustering, mRNA abundance, distance to nearest landmark site) can be found in Supplementary Figure S4. ( B ) Frequency profiles of longer than 6 nt homopolymeric tracks (poly dA (red), poly dT (blue)) around transcription landmark sites. ( C ) Average normalized profiles (top) and GC-content profiles (bottom) for euchromatic genes in T40 separated into four different mRNA abundance quartiles. Darker colours reflect higher mRNA abundance.

Techniques Used:

Dynamic nucleosome occupancy around transcriptional start sites during intraerythrocytic development. ( A ) Screenshot displaying absolute (gDNA-corrected MNase-seq coverage plots) or relative nucleosome occupancy (log2-ratio over T5) and steady-state mRNA expression (strand-specific RNA-seq). Region shown is chr2:470 000–490 000. Blue gene: forward strand; Red gene: reverse strand; Dynamic regions are highlighted pink, expressed downstream regions are highlighted green. ( B ) Observed (red) and 1000 randomized (transparent black) smoothed distributions (kernel density estimation) of Pearson correlations between the nucleosome-occupancy profile of upstream dynamically-occupied windows and the mRNA abundance profile of the downstream gene. Every black line shows the distribution of correlations for a single permutation of the time-point labels. ( C ) Observed (red) and expected (black, grey) distributions of distances between 3461 dynamically-occupied upstream windows (with anti-correlation between nucleosome-occupancy and mRNA abundance profile) and ATG of the downstream gene. Displayed are counts for 175 bp bins. The mean (black) and 95%-confidence intervals (grey) of the expected distribution were derived from 1000 randomizations of dynamic windows within the upstream regions. ( D ) Observed (red) and expected/randomized (black) smoothed distribution of distances of TSSs to the closest dynamic window.
Figure Legend Snippet: Dynamic nucleosome occupancy around transcriptional start sites during intraerythrocytic development. ( A ) Screenshot displaying absolute (gDNA-corrected MNase-seq coverage plots) or relative nucleosome occupancy (log2-ratio over T5) and steady-state mRNA expression (strand-specific RNA-seq). Region shown is chr2:470 000–490 000. Blue gene: forward strand; Red gene: reverse strand; Dynamic regions are highlighted pink, expressed downstream regions are highlighted green. ( B ) Observed (red) and 1000 randomized (transparent black) smoothed distributions (kernel density estimation) of Pearson correlations between the nucleosome-occupancy profile of upstream dynamically-occupied windows and the mRNA abundance profile of the downstream gene. Every black line shows the distribution of correlations for a single permutation of the time-point labels. ( C ) Observed (red) and expected (black, grey) distributions of distances between 3461 dynamically-occupied upstream windows (with anti-correlation between nucleosome-occupancy and mRNA abundance profile) and ATG of the downstream gene. Displayed are counts for 175 bp bins. The mean (black) and 95%-confidence intervals (grey) of the expected distribution were derived from 1000 randomizations of dynamic windows within the upstream regions. ( D ) Observed (red) and expected/randomized (black) smoothed distribution of distances of TSSs to the closest dynamic window.

Techniques Used: Expressing, RNA Sequencing Assay, Derivative Assay

15) 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

Representative log 2 AEI distributions: GAB2, GNB1L and DISC1 . Each pair of bars represents the gDNA-based (red) and cDNA-based (blue) log 2 AEI ratios from a single sample. cDNA-based log 2 AEI ratios are arranged in order from most- negative on the left to most-positive on the right, forming a distinct distribution of log 2 AEI ratios for each gene. The selected genes illustrate three common patterns of log 2 AEI population distributions: uniphasic, skewed and biphasic. As described in the text and in Additional file 4 , log 2 AEI distributions often contain useful information concerning the number, location and linkage of the regulatory genetic variants that produce AEI in each gene.
Figure Legend Snippet: Representative log 2 AEI distributions: GAB2, GNB1L and DISC1 . Each pair of bars represents the gDNA-based (red) and cDNA-based (blue) log 2 AEI ratios from a single sample. cDNA-based log 2 AEI ratios are arranged in order from most- negative on the left to most-positive on the right, forming a distinct distribution of log 2 AEI ratios for each gene. The selected genes illustrate three common patterns of log 2 AEI population distributions: uniphasic, skewed and biphasic. As described in the text and in Additional file 4 , log 2 AEI distributions often contain useful information concerning the number, location and linkage of the regulatory genetic variants that produce AEI in each gene.

Techniques Used:

16) Product Images from "Whole Genome Sequencing-Based Mapping and Candidate Identification of Mutations from Fixed Zebrafish Tissue"

Article Title: Whole Genome Sequencing-Based Mapping and Candidate Identification of Mutations from Fixed Zebrafish Tissue

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.117.300212

The fh227 allele is accurately mapped to chromosome 12 and egr2b using gDNA extracted from fixed tissue. Dorsal view of mbp expression in 5 dpf zebrafish using WISH and the mbp riboprobe shows phenotypically normal expression (purple) of mbp along the pLLNs (arrowheads) (A) compared to severely reduced mbp expression along the pLLN (arrowheads) in egr2b fh227/ fh227 mutants (B). (C) When the ratio of variant to reference alleles in the mutant pool is compared to the sibling pool and graphed across the whole genome for egr2b fh227 , a clear spike on chromosome 12 is observed (box). This spike indicates genotypic linkage to the trait used to sort the mutant and sibling pools. (D) When looking at chromosome 12, egr2b fh227 is linked to a single region centered ∼10 Mb (arrow). (E) Linkage map of the egr2b fh227 allele showing the 21 different homozygous, nonsynonymous, protein-coding SNPs in the single chromosome 12 region linked to the decreased mbp expression in the PNS which was used to sort the mutant and sibling pools. The single introduced stop is the mutation responsible for the egr2b fh227 mutant phenotype.
Figure Legend Snippet: The fh227 allele is accurately mapped to chromosome 12 and egr2b using gDNA extracted from fixed tissue. Dorsal view of mbp expression in 5 dpf zebrafish using WISH and the mbp riboprobe shows phenotypically normal expression (purple) of mbp along the pLLNs (arrowheads) (A) compared to severely reduced mbp expression along the pLLN (arrowheads) in egr2b fh227/ fh227 mutants (B). (C) When the ratio of variant to reference alleles in the mutant pool is compared to the sibling pool and graphed across the whole genome for egr2b fh227 , a clear spike on chromosome 12 is observed (box). This spike indicates genotypic linkage to the trait used to sort the mutant and sibling pools. (D) When looking at chromosome 12, egr2b fh227 is linked to a single region centered ∼10 Mb (arrow). (E) Linkage map of the egr2b fh227 allele showing the 21 different homozygous, nonsynonymous, protein-coding SNPs in the single chromosome 12 region linked to the decreased mbp expression in the PNS which was used to sort the mutant and sibling pools. The single introduced stop is the mutation responsible for the egr2b fh227 mutant phenotype.

Techniques Used: Expressing, Variant Assay, Mutagenesis

17) Product Images from "SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states"

Article Title: SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states

Journal: BMC Genomics

doi: 10.1186/s12864-018-4530-3

NGS library construction with alternatively sheared gDNA by using SALP method. a Schematic of library construction with the enzymatically digested and sonicated DNA by using SALP method. b Distribution of reads density of NGS library constructed with the Hind III-digested HepG2 gDNA and the density of predicted Hind III restriction sites in whole genome. c Distribution of reads density of NGS library constructed with the sonicated HepG2 gDNA. The information of other chromosomes was shown as Additional file 10 : Figure S6 and Additional file 11 : Figure S7
Figure Legend Snippet: NGS library construction with alternatively sheared gDNA by using SALP method. a Schematic of library construction with the enzymatically digested and sonicated DNA by using SALP method. b Distribution of reads density of NGS library constructed with the Hind III-digested HepG2 gDNA and the density of predicted Hind III restriction sites in whole genome. c Distribution of reads density of NGS library constructed with the sonicated HepG2 gDNA. The information of other chromosomes was shown as Additional file 10 : Figure S6 and Additional file 11 : Figure S7

Techniques Used: Next-Generation Sequencing, Sonication, Construct

18) Product Images from "DNA Methylome Profiling on the Infinium HumanMethylation450 Array from Limiting Quantities of Genomic DNA from a Single, Small Archived Bloodspot"

Article Title: DNA Methylome Profiling on the Infinium HumanMethylation450 Array from Limiting Quantities of Genomic DNA from a Single, Small Archived Bloodspot

Journal: Genetic Testing and Molecular Biomarkers

doi: 10.1089/gtmb.2017.0019

Correlation plots of DNA methylation profiles between replicates from high (750 ng) and low (25 ng) starting quantities of Jurkat cell gDNA. (A) Array β-values from 750 ng gDNA technical replicates (see Methods section for definition). (B) Array β-values from 25 ng replicates. (C) Array β-values from 750 ng replicate 1 compared with 25 ng replicate 2. The graphs depict 483,280 array positions present in all samples tested; ρ = correlation coefficient.
Figure Legend Snippet: Correlation plots of DNA methylation profiles between replicates from high (750 ng) and low (25 ng) starting quantities of Jurkat cell gDNA. (A) Array β-values from 750 ng gDNA technical replicates (see Methods section for definition). (B) Array β-values from 25 ng replicates. (C) Array β-values from 750 ng replicate 1 compared with 25 ng replicate 2. The graphs depict 483,280 array positions present in all samples tested; ρ = correlation coefficient.

Techniques Used: DNA Methylation Assay

Reproducibility of DNA methylation profiles from 25 ng of gDNA isolated from small archived dried bloodspots. (A) Correlation plot of array β-values from 25 ng replicate samples. (B) Correlation plot of array β-values between two independent samples representing the median correlation coefficient (0.9873) from all pair-wise comparisons ( n = 190) of 20 individuals. (C) Distribution of the standard deviations of mean β-values obtained from averaging array loci data from 20 individuals.
Figure Legend Snippet: Reproducibility of DNA methylation profiles from 25 ng of gDNA isolated from small archived dried bloodspots. (A) Correlation plot of array β-values from 25 ng replicate samples. (B) Correlation plot of array β-values between two independent samples representing the median correlation coefficient (0.9873) from all pair-wise comparisons ( n = 190) of 20 individuals. (C) Distribution of the standard deviations of mean β-values obtained from averaging array loci data from 20 individuals.

Techniques Used: DNA Methylation Assay, Isolation

Signal detection attributes of array loci using different starting quantities (25, 100, and 750 ng) of Jurkat cell genomic DNA (gDNA). (A) The cumulative distributions of detection p -values for the subset of array loci with p > 5 × 10 −6 . (B) The distributions of raw signal intensities for 483,280 array positions present at all starting DNA concentrations. (C) Pair-wise comparison of the signal intensities in the 25 and 750 ng samples of the loci depicted in (B) .
Figure Legend Snippet: Signal detection attributes of array loci using different starting quantities (25, 100, and 750 ng) of Jurkat cell genomic DNA (gDNA). (A) The cumulative distributions of detection p -values for the subset of array loci with p > 5 × 10 −6 . (B) The distributions of raw signal intensities for 483,280 array positions present at all starting DNA concentrations. (C) Pair-wise comparison of the signal intensities in the 25 and 750 ng samples of the loci depicted in (B) .

Techniques Used:

19) Product Images from "The house fly Y Chromosome is young and minimally differentiated from its ancient X Chromosome partner"

Article Title: The house fly Y Chromosome is young and minimally differentiated from its ancient X Chromosome partner

Journal: Genome Research

doi: 10.1101/gr.215509.116

There is elevated heterozygosity on the third chromosome in III M males, but not on the X Chromosome in XY males. Box plots show the distribution of the percentage of heterozygous SNPs within genes on each chromosome in either XY aabys males relative to XX aabys females using genomic DNA sequences ( A ) or III M males relative to XY males using RNA-seq data ( B ). Values > 50% indicate elevated heterozygosity in XY males or III M males. The median across all autosomes is indicated by a dashed line.
Figure Legend Snippet: There is elevated heterozygosity on the third chromosome in III M males, but not on the X Chromosome in XY males. Box plots show the distribution of the percentage of heterozygous SNPs within genes on each chromosome in either XY aabys males relative to XX aabys females using genomic DNA sequences ( A ) or III M males relative to XY males using RNA-seq data ( B ). Values > 50% indicate elevated heterozygosity in XY males or III M males. The median across all autosomes is indicated by a dashed line.

Techniques Used: RNA Sequencing Assay

20) Product Images from "Fragmentation Through Polymerization (FTP): A new method to fragment DNA for next-generation sequencing"

Article Title: Fragmentation Through Polymerization (FTP): A new method to fragment DNA for next-generation sequencing

Journal: PLoS ONE

doi: 10.1371/journal.pone.0210374

Agarose-gel electrophoresis of gDNA fragmented by the FTP method. gDNA of E . coli BL21 was incubated as described in Materials and Methods: without enzymes (lane 1), with SD polymerase (lane 2), with DNase I (lane 3), and with both DNase I and SD polymerase (lane 4 and 5). M1: 1 kb DNA Ladder; M2: 100 bp DNA Ladder.
Figure Legend Snippet: Agarose-gel electrophoresis of gDNA fragmented by the FTP method. gDNA of E . coli BL21 was incubated as described in Materials and Methods: without enzymes (lane 1), with SD polymerase (lane 2), with DNase I (lane 3), and with both DNase I and SD polymerase (lane 4 and 5). M1: 1 kb DNA Ladder; M2: 100 bp DNA Ladder.

Techniques Used: Agarose Gel Electrophoresis, Incubation

21) Product Images from "Data analysis issues for allele-specific expression using Illumina's GoldenGate assay"

Article Title: Data analysis issues for allele-specific expression using Illumina's GoldenGate assay

Journal: BMC Bioinformatics

doi: 10.1186/1471-2105-11-280

Intensity trends in slope for the regression between the cDNA and gDNA log-ratios where average intensity has been ascertained using a different microarray platform . Slopes from the regression of cDNA log-ratios against gDNA log-ratios from the 44 CEU individuals in Tan et al . (2008) for SNPs with at least 3 individuals of each homozygous genotype (AA, BB) versus average intensity measured on the Illumina WG-6 arrays from Stranger et al . (2007) are shown. In panel 1, 155 SNPs are plotted and in panel 2, 152 SNPs are shown. As observed in Figure 7, we see a trend for increasing slope with increasing intensity.
Figure Legend Snippet: Intensity trends in slope for the regression between the cDNA and gDNA log-ratios where average intensity has been ascertained using a different microarray platform . Slopes from the regression of cDNA log-ratios against gDNA log-ratios from the 44 CEU individuals in Tan et al . (2008) for SNPs with at least 3 individuals of each homozygous genotype (AA, BB) versus average intensity measured on the Illumina WG-6 arrays from Stranger et al . (2007) are shown. In panel 1, 155 SNPs are plotted and in panel 2, 152 SNPs are shown. As observed in Figure 7, we see a trend for increasing slope with increasing intensity.

Techniques Used: Microarray

Intensity trends in slope and intercept for the regression analysis between the cDNA and gDNA log-ratios . The slopes and intercepts were calculated from the data in Tan et al . (2008) for SNPs with at least 3 AA and 3 BB individuals from the complete set of 142 samples. The average intensity for each SNP (calculated using the same data) is plotted on the x-axis. In total 277 SNPs are plotted from panel 1 and 261 from panel 2. Log-ratios were calculated after within-array quantile normalization of the Cy5 and Cy3 intensities. This figure shows an increasing trend for slope as average intensity increases. There is no such trend for intercept.
Figure Legend Snippet: Intensity trends in slope and intercept for the regression analysis between the cDNA and gDNA log-ratios . The slopes and intercepts were calculated from the data in Tan et al . (2008) for SNPs with at least 3 AA and 3 BB individuals from the complete set of 142 samples. The average intensity for each SNP (calculated using the same data) is plotted on the x-axis. In total 277 SNPs are plotted from panel 1 and 261 from panel 2. Log-ratios were calculated after within-array quantile normalization of the Cy5 and Cy3 intensities. This figure shows an increasing trend for slope as average intensity increases. There is no such trend for intercept.

Techniques Used:

Quantification of dye effects . Smoothed scatter plots of the estimated dye effect from the SNP-wise linear models for the non-normalized gDNA (A) and cDNA (B) data versus average intensity are shown. In these plots, a higher density of points is represented by a darker shade of blue. For each analysis, the log-ratios are calculated as log 2 ( Cy 5/ Cy 3). Probes at lower intensities tend to have a bias towards the Cy3 channel (negative dye effect), while probes with higher intensities generally have a bias towards the Cy5 channel (positive dye-bias). Panel C shows the estimated dye effect before and after quantile normalization. The dye effect is systematically larger, and more variable before quantile normalization for both sample types. After quantile normalization, the dye effects are centered around zero.
Figure Legend Snippet: Quantification of dye effects . Smoothed scatter plots of the estimated dye effect from the SNP-wise linear models for the non-normalized gDNA (A) and cDNA (B) data versus average intensity are shown. In these plots, a higher density of points is represented by a darker shade of blue. For each analysis, the log-ratios are calculated as log 2 ( Cy 5/ Cy 3). Probes at lower intensities tend to have a bias towards the Cy3 channel (negative dye effect), while probes with higher intensities generally have a bias towards the Cy5 channel (positive dye-bias). Panel C shows the estimated dye effect before and after quantile normalization. The dye effect is systematically larger, and more variable before quantile normalization for both sample types. After quantile normalization, the dye effects are centered around zero.

Techniques Used:

Plots of cDNA versus gDNA log-ratios for 3 SNPs located in known imprinted genes . Averaged log-ratios from the 44 CEU samples in Tan et al . (2008) are shown. The first example (PEG10) provides a clear example of ASE, with the AB cDNA log-ratios (red) at similar levels to either the AA, of BB cDNA log-ratios, which is indicative of silencing. For this SNP, there is a clear linear trend between the homozygous cDNA and gDNA log-ratios. In the second example (IGF2R), there is again a linear trend, although in this case, there is no evidence for ASE, with the AB log-ratios around zero. In the final example (GABRG3), there is no obvious linear trend. This SNP provides an example of non-specific signal, where the cDNA log-ratios lie around zero irrespective of the alleles present.
Figure Legend Snippet: Plots of cDNA versus gDNA log-ratios for 3 SNPs located in known imprinted genes . Averaged log-ratios from the 44 CEU samples in Tan et al . (2008) are shown. The first example (PEG10) provides a clear example of ASE, with the AB cDNA log-ratios (red) at similar levels to either the AA, of BB cDNA log-ratios, which is indicative of silencing. For this SNP, there is a clear linear trend between the homozygous cDNA and gDNA log-ratios. In the second example (IGF2R), there is again a linear trend, although in this case, there is no evidence for ASE, with the AB log-ratios around zero. In the final example (GABRG3), there is no obvious linear trend. This SNP provides an example of non-specific signal, where the cDNA log-ratios lie around zero irrespective of the alleles present.

Techniques Used:

Various plots of the raw signal from GoldenGate arrays measuring ASE . Panels A and B show boxplots of the summarized log 2 ( Cy 5) and log 2 ( Cy 3) intensities respectively from a representative SAM. The data from each array were plotted in a separate boxplot, and color coded by sample (blue - gDNA, red - cDNA). Arrays 4 and 7 have low signal in both channels (IQR ≤ 1) and were excluded from downstream analysis. Density plots for each channel from two typical gDNA and cDNA arrays are presented in panels C and D respectively. These plots also show the systematic difference in overall signal between gDNA and cDNA hybridizations. Smoothed MA -plots for the gDNA (E) and cDNA (F) also highlight the differences. In these plots, a higher density of points is represented by a darker shade of blue.
Figure Legend Snippet: Various plots of the raw signal from GoldenGate arrays measuring ASE . Panels A and B show boxplots of the summarized log 2 ( Cy 5) and log 2 ( Cy 3) intensities respectively from a representative SAM. The data from each array were plotted in a separate boxplot, and color coded by sample (blue - gDNA, red - cDNA). Arrays 4 and 7 have low signal in both channels (IQR ≤ 1) and were excluded from downstream analysis. Density plots for each channel from two typical gDNA and cDNA arrays are presented in panels C and D respectively. These plots also show the systematic difference in overall signal between gDNA and cDNA hybridizations. Smoothed MA -plots for the gDNA (E) and cDNA (F) also highlight the differences. In these plots, a higher density of points is represented by a darker shade of blue.

Techniques Used:

22) Product Images from "Characterisation of the changing genomic landscape of metastatic melanoma using cell free DNA"

Article Title: Characterisation of the changing genomic landscape of metastatic melanoma using cell free DNA

Journal: NPJ genomic medicine

doi: 10.1038/s41525-017-0030-7

Coverage uniformity of WGS libraries. a Represented is the cumulative proportion of sequencing coverage per cumulative proportion of sequence in whole genome sequencing across normal germline DNA (gDNA) from peripheral blood mononuclear cells, two cfDNA time points, and an archival FFPE tissue biopsy from a metastatic melanoma patient. If coverage was perfectly uniform across the genome coverage the relationship would be linear with gradient one. b Mapped depth of coverage distribution for WGS sequencing runs. The range of the coverage distribution is truncated at 150. Each trace is annotated with the mode of the distribution. c Insert size distribution of sequencing reads for the sequencing runs. The distribution is truncated at 300 base pairs. Each trace is annotated with the mode of the distribution
Figure Legend Snippet: Coverage uniformity of WGS libraries. a Represented is the cumulative proportion of sequencing coverage per cumulative proportion of sequence in whole genome sequencing across normal germline DNA (gDNA) from peripheral blood mononuclear cells, two cfDNA time points, and an archival FFPE tissue biopsy from a metastatic melanoma patient. If coverage was perfectly uniform across the genome coverage the relationship would be linear with gradient one. b Mapped depth of coverage distribution for WGS sequencing runs. The range of the coverage distribution is truncated at 150. Each trace is annotated with the mode of the distribution. c Insert size distribution of sequencing reads for the sequencing runs. The distribution is truncated at 300 base pairs. Each trace is annotated with the mode of the distribution

Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded

23) Product Images from "Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape"

Article Title: Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape

Journal: Cell

doi: 10.1016/j.cell.2016.04.038

The ARF Family Preferentially Binds to Phased Motif Clusters that Are Enriched in Target Gene Promoters (A) Three possible orientations of an ARF motif repeat. ). (C) Relative frequencies of DAP-seq peaks at DR, ER, and IR pairs for Arabidopsis (AtARF5 and AtARF2) and maize (ARF5/ZmARF29) proteins interrogating Arabidopsis (At-gDNA) or maize (Zm-gDNA) DAP-seq libraries. (D) A cluster of 13 phased TGTC sites (red ticks) in the promoter of the ARF5 target IAA5. Black ticks are non-phased TGTC sites. (E) DAP-seq signal at the TSS (x axis) of ARF5 direct target genes, non-auxin-responsive background genes, and all genes. .
Figure Legend Snippet: The ARF Family Preferentially Binds to Phased Motif Clusters that Are Enriched in Target Gene Promoters (A) Three possible orientations of an ARF motif repeat. ). (C) Relative frequencies of DAP-seq peaks at DR, ER, and IR pairs for Arabidopsis (AtARF5 and AtARF2) and maize (ARF5/ZmARF29) proteins interrogating Arabidopsis (At-gDNA) or maize (Zm-gDNA) DAP-seq libraries. (D) A cluster of 13 phased TGTC sites (red ticks) in the promoter of the ARF5 target IAA5. Black ticks are non-phased TGTC sites. (E) DAP-seq signal at the TSS (x axis) of ARF5 direct target genes, non-auxin-responsive background genes, and all genes. .

Techniques Used:

Genome-wide TFBS Discovery by DAP-Seq (A) Preparation of DAP- and ampDAP-seq libraries. (B) Expression and capture of affinity-tagged TFs. (C) gDNA is bound to immobilized TFs, eluted, and sequenced. (D) ABI5 DAP- and ChIP-seq peaks at a known regulatory element in the ABI5 promoter. ). .
Figure Legend Snippet: Genome-wide TFBS Discovery by DAP-Seq (A) Preparation of DAP- and ampDAP-seq libraries. (B) Expression and capture of affinity-tagged TFs. (C) gDNA is bound to immobilized TFs, eluted, and sequenced. (D) ABI5 DAP- and ChIP-seq peaks at a known regulatory element in the ABI5 promoter. ). .

Techniques Used: Genome Wide, Expressing, Chromatin Immunoprecipitation

24) Product Images from "Extreme MHC class I diversity in the sedge warbler (Acrocephalus schoenobaenus); selection patterns and allelic divergence suggest that different genes have different functions"

Article Title: Extreme MHC class I diversity in the sedge warbler (Acrocephalus schoenobaenus); selection patterns and allelic divergence suggest that different genes have different functions

Journal: BMC Evolutionary Biology

doi: 10.1186/s12862-017-0997-9

Neighbor-net network of sedge warbler MHC class I exon 3 alleles constructed from all cDNA and gDNA alleles detected in four individuals, alleles marked with bold and italics were found only in gDNA amplicons. Bootstrap support values (based on 1000 replicates) higher than 70% are presented. The loops imply areas of phylogenetic uncertainty or reticulations. 3 bp deletion alleles are shown in orange , 6 bp deletion alleles in green and alleles with no deletion in blue
Figure Legend Snippet: Neighbor-net network of sedge warbler MHC class I exon 3 alleles constructed from all cDNA and gDNA alleles detected in four individuals, alleles marked with bold and italics were found only in gDNA amplicons. Bootstrap support values (based on 1000 replicates) higher than 70% are presented. The loops imply areas of phylogenetic uncertainty or reticulations. 3 bp deletion alleles are shown in orange , 6 bp deletion alleles in green and alleles with no deletion in blue

Techniques Used: Construct

25) Product Images from "A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes"

Article Title: A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes

Journal: Genome Research

doi: 10.1101/gr.176115.114

UBTF1/2 binds histone genes. ( A–C ) IGV (Integrated Genome Viewer) screenshots of mapped reads from UBTF1/2 ChIP and input gDNA at mouse histone gene cluster 1 ( A ), cluster 2 ( B ), and histone variant genes H2afx and H3f3a ( C ) in NIH3T3 cells. (
Figure Legend Snippet: UBTF1/2 binds histone genes. ( A–C ) IGV (Integrated Genome Viewer) screenshots of mapped reads from UBTF1/2 ChIP and input gDNA at mouse histone gene cluster 1 ( A ), cluster 2 ( B ), and histone variant genes H2afx and H3f3a ( C ) in NIH3T3 cells. (

Techniques Used: Chromatin Immunoprecipitation, Variant Assay

26) Product Images from "Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing"

Article Title: Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing

Journal: Plant Methods

doi: 10.1186/s13007-018-0336-1

A typical DNA extraction from freeze-dried and fresh tissue using the 96-well plate method. Genomic DNA was extracted from 96 individuals of a Trifolium repens (white clover; freeze-dried leaf) and b Lolium perenne ). One T. repens individual had a poor DNA yield (top row, ninth lane from the right) and was subsequently re-extracted using the Individual Tube freeze-dried tissue protocol. These DNA samples have been used for developing genotyping-by-sequencing libraries and yielded high-quality sequence data
Figure Legend Snippet: A typical DNA extraction from freeze-dried and fresh tissue using the 96-well plate method. Genomic DNA was extracted from 96 individuals of a Trifolium repens (white clover; freeze-dried leaf) and b Lolium perenne ). One T. repens individual had a poor DNA yield (top row, ninth lane from the right) and was subsequently re-extracted using the Individual Tube freeze-dried tissue protocol. These DNA samples have been used for developing genotyping-by-sequencing libraries and yielded high-quality sequence data

Techniques Used: DNA Extraction, Sequencing

] was modified for gDNA extraction using 96-well plates ( a ] to a 96-well plate protocol ( b ), and development of a streamlined inexpensive protocol described in this paper ( c ] comprising 95 individuals from each of the white clover gDNA extractions shown above ( a – c ] for GBS libraries made from the gDNA shown a – c is represented in graphs describing quality across all bases from every sequence read at each position ( d – f , respectively). Sequence quality is based on phred ], an exponential scale where, for example, 20 = one incorrect sequence base-call in 100, and 30 = one incorrect base-call in 1000. The y-axis shows the quality scores, and the higher the score the greater confidence in the base-calls at that position. The background of the graph divides the y-axis into very good quality calls (green), reasonable quality (orange), and poor quality (red)
Figure Legend Snippet: ] was modified for gDNA extraction using 96-well plates ( a ] to a 96-well plate protocol ( b ), and development of a streamlined inexpensive protocol described in this paper ( c ] comprising 95 individuals from each of the white clover gDNA extractions shown above ( a – c ] for GBS libraries made from the gDNA shown a – c is represented in graphs describing quality across all bases from every sequence read at each position ( d – f , respectively). Sequence quality is based on phred ], an exponential scale where, for example, 20 = one incorrect sequence base-call in 100, and 30 = one incorrect base-call in 1000. The y-axis shows the quality scores, and the higher the score the greater confidence in the base-calls at that position. The background of the graph divides the y-axis into very good quality calls (green), reasonable quality (orange), and poor quality (red)

Techniques Used: Modification, Sequencing

27) Product Images from "A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes"

Article Title: A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes

Journal: Genome Research

doi: 10.1101/gr.176115.114

UBTF1/2 binds histone genes. ( A–C ) IGV (Integrated Genome Viewer) screenshots of mapped reads from UBTF1/2 ChIP and input gDNA at mouse histone gene cluster 1 ( A ), cluster 2 ( B ), and histone variant genes H2afx and H3f3a ( C ) in NIH3T3 cells. (
Figure Legend Snippet: UBTF1/2 binds histone genes. ( A–C ) IGV (Integrated Genome Viewer) screenshots of mapped reads from UBTF1/2 ChIP and input gDNA at mouse histone gene cluster 1 ( A ), cluster 2 ( B ), and histone variant genes H2afx and H3f3a ( C ) in NIH3T3 cells. (

Techniques Used: Chromatin Immunoprecipitation, Variant Assay

28) Product Images from "High Quality Genome-Wide Genotyping from Archived Dried Blood Spots without DNA Amplification"

Article Title: High Quality Genome-Wide Genotyping from Archived Dried Blood Spots without DNA Amplification

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064710

Genotyping performance as a function of gDNA concentration. The genotyping performance of the gDNA samples (A) and wgaDNA samples (B) described in Figure 1 is plotted against the DNA concentration of the sample as determined by Qubit for gDNA and Picogreen for wgaDNA (bottom axis) and total amount of DNA used (4 µl) during each genotyping run (top axis). Picogreen was used for wgaDNA because Qubit does not discern concentrations above 100 ng/µl. Note that all gDNA samples with DNA concentrations above 5 ng/µl (∼20 ng DNA) were genotyped successfully (A). No similar threshold was observed for wgaDNA samples (B).
Figure Legend Snippet: Genotyping performance as a function of gDNA concentration. The genotyping performance of the gDNA samples (A) and wgaDNA samples (B) described in Figure 1 is plotted against the DNA concentration of the sample as determined by Qubit for gDNA and Picogreen for wgaDNA (bottom axis) and total amount of DNA used (4 µl) during each genotyping run (top axis). Picogreen was used for wgaDNA because Qubit does not discern concentrations above 100 ng/µl. Note that all gDNA samples with DNA concentrations above 5 ng/µl (∼20 ng DNA) were genotyped successfully (A). No similar threshold was observed for wgaDNA samples (B).

Techniques Used: Concentration Assay

Reproducibility of gDNA yields from the same dried blood spot. gDNA was extracted from two 2 mm punches of seven different archived DBS (1–7), and the process was repeated with fresh punches from the same DBS on a separate day. DNA was quantitated by Qubit. Different DBS showed ∼5-fold difference in DNA yield, whereas extraction duplicates of the same DBS gave almost identical DNA yield (103% ±15%, mean ± S.D.).
Figure Legend Snippet: Reproducibility of gDNA yields from the same dried blood spot. gDNA was extracted from two 2 mm punches of seven different archived DBS (1–7), and the process was repeated with fresh punches from the same DBS on a separate day. DNA was quantitated by Qubit. Different DBS showed ∼5-fold difference in DNA yield, whereas extraction duplicates of the same DBS gave almost identical DNA yield (103% ±15%, mean ± S.D.).

Techniques Used:

gDNA yield increases with increasing number of dried blood spot punches. The indicated number of punches was obtained from 19 archived DBS for gDNA extraction. DNA yield was determined by Qubit ( Table S1 ) and the values (mean ± S.D.) were normalized to the yield for a single punch. p
Figure Legend Snippet: gDNA yield increases with increasing number of dried blood spot punches. The indicated number of punches was obtained from 19 archived DBS for gDNA extraction. DNA yield was determined by Qubit ( Table S1 ) and the values (mean ± S.D.) were normalized to the yield for a single punch. p

Techniques Used:

Success and accuracy of genotyping with genomic or amplified DNA from dried blood spots. Genomic DNA (gDNA) extracted from two 2 mm punches obtained from archived dried blood spots (DBS) from nine individuals (1–9) was genotyped on an Illumina Infinium 300,000 SNP test chip (set “a”, 1a–9a). For six of the DBS (1,3,4,5,7,8), a set (set “b”) of extraction duplicates (duplicate extractions from the same DBS) was prepared and genotyped in the same way. A 1 µl aliquot of each of these 15 samples of gDNA was whole genome amplified (wgaDNA) and then genotyped in the same way as sets “a” and “b”: set “c” wgaDNA samples (1c–9c) were amplified from set “a” gDNA samples, and set “d” wgaDNA samples (1d, 3d, 4d, 5d, 7d, 8d) were amplified from set “b” gDNA samples. (A) Performance quality of the gDNA and wgaDNA samples on genome-wide SNP genotyping. Successful, Marginal, and Failed sample performance parameters are given in Results. (B) SNP genotyping replication error. The number of SNP discrepancies for each sample was determined using gDNA set “a” as the reference. Replication error (the number of discrepancies among SNPs called in both the sample and reference divided by the total number of SNPs called in both) was 1.6×10 −5 for set “b”, 1.5×10 −4 for set “c”, and 1.2×10 −4 for set “d”. These replication error values are provisional because we do not know the true genotype of the discrepant loci and there may be gDNA-specific and/or wgaDNA-specific artifacts.
Figure Legend Snippet: Success and accuracy of genotyping with genomic or amplified DNA from dried blood spots. Genomic DNA (gDNA) extracted from two 2 mm punches obtained from archived dried blood spots (DBS) from nine individuals (1–9) was genotyped on an Illumina Infinium 300,000 SNP test chip (set “a”, 1a–9a). For six of the DBS (1,3,4,5,7,8), a set (set “b”) of extraction duplicates (duplicate extractions from the same DBS) was prepared and genotyped in the same way. A 1 µl aliquot of each of these 15 samples of gDNA was whole genome amplified (wgaDNA) and then genotyped in the same way as sets “a” and “b”: set “c” wgaDNA samples (1c–9c) were amplified from set “a” gDNA samples, and set “d” wgaDNA samples (1d, 3d, 4d, 5d, 7d, 8d) were amplified from set “b” gDNA samples. (A) Performance quality of the gDNA and wgaDNA samples on genome-wide SNP genotyping. Successful, Marginal, and Failed sample performance parameters are given in Results. (B) SNP genotyping replication error. The number of SNP discrepancies for each sample was determined using gDNA set “a” as the reference. Replication error (the number of discrepancies among SNPs called in both the sample and reference divided by the total number of SNPs called in both) was 1.6×10 −5 for set “b”, 1.5×10 −4 for set “c”, and 1.2×10 −4 for set “d”. These replication error values are provisional because we do not know the true genotype of the discrepant loci and there may be gDNA-specific and/or wgaDNA-specific artifacts.

Techniques Used: Amplification, Chromatin Immunoprecipitation, Genome Wide

29) Product Images from "Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis"

Article Title: Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0178706

Automated high throughput FormaPure-based extraction protocol. ( A ) Work flow illustration of sample acquisition, upstream sample processing and extraction. Note that a separate high temperature incubation step is added to facilitate the reversal of remaining crosslinks. The upstream processes are manual in the original protocol whereas those steps are modified to be suitable for automation in the modified protocol. The in-house on-deck heating blocks were instrumental in rendering the lysis/deparaffinization steps automatable. Acquisition of samples in SBS format matrix tubes with their automated capping and decapping were also further measures that allowed the entire process to be amenable for automated liquid handling. ( B ) gDNA yield. Historical gDNA yield data from the Qiagen/High Pure protocol (Q; n = 142) using equivalent sizes of numerous FFPE samples of lymphoma origin was compared with that of the FormaPure protocol (F; n-91). (C) RNA yield. Comparison of the Qiagen-High Pure (Q-H), and FormaPure (F) protocols are shown. N = 142 for Q-H and N = 44 for F.
Figure Legend Snippet: Automated high throughput FormaPure-based extraction protocol. ( A ) Work flow illustration of sample acquisition, upstream sample processing and extraction. Note that a separate high temperature incubation step is added to facilitate the reversal of remaining crosslinks. The upstream processes are manual in the original protocol whereas those steps are modified to be suitable for automation in the modified protocol. The in-house on-deck heating blocks were instrumental in rendering the lysis/deparaffinization steps automatable. Acquisition of samples in SBS format matrix tubes with their automated capping and decapping were also further measures that allowed the entire process to be amenable for automated liquid handling. ( B ) gDNA yield. Historical gDNA yield data from the Qiagen/High Pure protocol (Q; n = 142) using equivalent sizes of numerous FFPE samples of lymphoma origin was compared with that of the FormaPure protocol (F; n-91). (C) RNA yield. Comparison of the Qiagen-High Pure (Q-H), and FormaPure (F) protocols are shown. N = 142 for Q-H and N = 44 for F.

Techniques Used: High Throughput Screening Assay, Flow Cytometry, Incubation, Modification, Lysis, Formalin-fixed Paraffin-Embedded

30) Product Images from "Genome and metagenome sequencing: Using the human methyl-binding domain to partition genomic DNA derived from plant tissues 1"

Article Title: Genome and metagenome sequencing: Using the human methyl-binding domain to partition genomic DNA derived from plant tissues 1

Journal: Applications in Plant Sciences

doi: 10.3732/apps.1400064

Arabidopsis thaliana read-pairs mapping to any of the three genomes (mitochondrial DNA [mtDNA], chloroplast DNA [cpDNA], and nuclear DNA [nDNA]). UT = untreated genomic DNA (gDNA) library; E = methyl-enriched gDNA library; D = methyl-depleted gDNA library.
Figure Legend Snippet: Arabidopsis thaliana read-pairs mapping to any of the three genomes (mitochondrial DNA [mtDNA], chloroplast DNA [cpDNA], and nuclear DNA [nDNA]). UT = untreated genomic DNA (gDNA) library; E = methyl-enriched gDNA library; D = methyl-depleted gDNA library.

Techniques Used:

31) Product Images from "GSK3β is a key regulator of the ROS-dependent necrotic death induced by the quinone DMNQ"

Article Title: GSK3β is a key regulator of the ROS-dependent necrotic death induced by the quinone DMNQ

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-2202-0

Screening protocol and targets validation. a Schematic representation of the screening protocol. 66 × 10 6 millions of cells were infected with the lentiviral particles containing the shRNA plasmids with a MOI of 0,1 for 2 days. For the selection of the expressing-shRNAs, cells were first grown in the presence of puromycin (2 μg/ml) for additional 3 days and subsequently treated with G5 (2.5 μM) for 60 h. Surviving cells were harvested to recover the genomic DNA. b Representation of the most enriched hits. Data are represented as fold enrichment respect to the median of all shRNAs. The enrichment is relative to the second most abundant shRNA. Only shRNA with a fold increase > 3 are shown. c Hits that are in relationship with GSK3β as up-stream regulators or downstream effectors. d U87MG cells were silenced for CAPN1 and PP2AC and, after 48 h from transfection, they were treated with G5 (2.5 μM) for 24 h. Cell death was calculated as percentage of cells positive to PI staining using cytofluorimetric analysis. Data are from three experiments; +SD. e Cellular lysates from the silenced cells were analyzed by immunoblot. Antibodies anti-CAPN1, anti-PP2AC, and anti-Actin (as loading control) were used as indicated. f U87MG and U87MG-BCLXL cells were silenced for GSK3β. After 48 h they were treated with G5 (2.5 μM) for further 24 h. Cell death was calculated as percentage of cells positive to PI staining using cytofluorimetric analysis. Data are from three experiments; +SD. g qRT-PCR analysis of GSK3β mRNA levels in silenced cells. Data were from three experiments; +SD. h Immunoblot analysis of GSK3β levels in the indicated clones of U87MG cells, selected after CRISPR/Cas9 mediated knock-out. Actin was used as loading control. i Cytofluorimetric analysis were performed to measure the % of PI positivity in the different U87MG clones treated with G5 (10 µM) for 24 h. Data are presented as mean ± SD. n = 3.
Figure Legend Snippet: Screening protocol and targets validation. a Schematic representation of the screening protocol. 66 × 10 6 millions of cells were infected with the lentiviral particles containing the shRNA plasmids with a MOI of 0,1 for 2 days. For the selection of the expressing-shRNAs, cells were first grown in the presence of puromycin (2 μg/ml) for additional 3 days and subsequently treated with G5 (2.5 μM) for 60 h. Surviving cells were harvested to recover the genomic DNA. b Representation of the most enriched hits. Data are represented as fold enrichment respect to the median of all shRNAs. The enrichment is relative to the second most abundant shRNA. Only shRNA with a fold increase > 3 are shown. c Hits that are in relationship with GSK3β as up-stream regulators or downstream effectors. d U87MG cells were silenced for CAPN1 and PP2AC and, after 48 h from transfection, they were treated with G5 (2.5 μM) for 24 h. Cell death was calculated as percentage of cells positive to PI staining using cytofluorimetric analysis. Data are from three experiments; +SD. e Cellular lysates from the silenced cells were analyzed by immunoblot. Antibodies anti-CAPN1, anti-PP2AC, and anti-Actin (as loading control) were used as indicated. f U87MG and U87MG-BCLXL cells were silenced for GSK3β. After 48 h they were treated with G5 (2.5 μM) for further 24 h. Cell death was calculated as percentage of cells positive to PI staining using cytofluorimetric analysis. Data are from three experiments; +SD. g qRT-PCR analysis of GSK3β mRNA levels in silenced cells. Data were from three experiments; +SD. h Immunoblot analysis of GSK3β levels in the indicated clones of U87MG cells, selected after CRISPR/Cas9 mediated knock-out. Actin was used as loading control. i Cytofluorimetric analysis were performed to measure the % of PI positivity in the different U87MG clones treated with G5 (10 µM) for 24 h. Data are presented as mean ± SD. n = 3.

Techniques Used: Infection, shRNA, Selection, Expressing, Transfection, Staining, Quantitative RT-PCR, Clone Assay, CRISPR, Knock-Out

32) Product Images from "Azacitidine combined with the selective FLT3 kinase inhibitor crenolanib disrupts stromal protection and inhibits expansion of residual leukemia-initiating cells in FLT3-ITD AML with concurrent epigenetic mutations"

Article Title: Azacitidine combined with the selective FLT3 kinase inhibitor crenolanib disrupts stromal protection and inhibits expansion of residual leukemia-initiating cells in FLT3-ITD AML with concurrent epigenetic mutations

Journal: Oncotarget

doi: 10.18632/oncotarget.21877

Detection of FLT3 -ITD and concurrent gene mutations in leukemic stem/progenitor compartments Experimental design (A) . Gating strategy for multiparameter flow cytometric sorting of CD34 + and CD34 - AML BM samples. Blasts (Lin - /CD33 + /CD34 - ), committed progenitors (Lin - /CD33 (+) /CD45 dim /CD34 + CD38 + ), and early stem cell compartments (Lin - /CD33 (+) /CD45 dim /CD34 + CD38 - ) i.e. MLP (CD45RA + ), MPP (CD45RA - CD90 - ) and HSC (CD45RA - CD90 + ). Representative plots for a CD34 + AML (upper panel, Table 1 patient # 2) and CD34 - AML sample (lower panel, Table 1 patient # 7) are shown (B) . gDNA was isolated from sorted compartments and mutations in FLT3 , NPM1 , DNMT3A , TET2 and IDH1/2 genes were detected by targeted resequencing. Variant allele frequencies (VAF, %) of FLT3 -ITD ( C , upper panel) and epigenetic mutations (C, lower panel) in sorted populations are shown. n.a., not available (e.g. population not found).
Figure Legend Snippet: Detection of FLT3 -ITD and concurrent gene mutations in leukemic stem/progenitor compartments Experimental design (A) . Gating strategy for multiparameter flow cytometric sorting of CD34 + and CD34 - AML BM samples. Blasts (Lin - /CD33 + /CD34 - ), committed progenitors (Lin - /CD33 (+) /CD45 dim /CD34 + CD38 + ), and early stem cell compartments (Lin - /CD33 (+) /CD45 dim /CD34 + CD38 - ) i.e. MLP (CD45RA + ), MPP (CD45RA - CD90 - ) and HSC (CD45RA - CD90 + ). Representative plots for a CD34 + AML (upper panel, Table 1 patient # 2) and CD34 - AML sample (lower panel, Table 1 patient # 7) are shown (B) . gDNA was isolated from sorted compartments and mutations in FLT3 , NPM1 , DNMT3A , TET2 and IDH1/2 genes were detected by targeted resequencing. Variant allele frequencies (VAF, %) of FLT3 -ITD ( C , upper panel) and epigenetic mutations (C, lower panel) in sorted populations are shown. n.a., not available (e.g. population not found).

Techniques Used: Flow Cytometry, Isolation, Variant Assay

33) Product Images from "An ENU-induced splicing mutation reveals a role for Unc93b1 in early immune cell activation following Influenza A H1N1 infection"

Article Title: An ENU-induced splicing mutation reveals a role for Unc93b1 in early immune cell activation following Influenza A H1N1 infection

Journal: Genes and immunity

doi: 10.1038/gene.2014.22

The Letr allele causes a single nucleotide transversion and alternative splicing of Unc93b1 (A) Chromatogram of the Unc93b1 +/+ (top) and Unc93b1 Letr/Letr (bottom) gDNA sequence with the single T to A nucleotide change caused by the Letr mutation that is identified on the non-coding strand as A to T. (B) Gel electrophoresis of cDNA from Unc93b1 Letr/Letr , Unc93b1 +/Letr , and wild type inbred strains (C57BL/6, 129S1, C3H/HeN). The wild type transcript size is 918bp while the mutant transcript size is 756bp. (C) Schematic of Unc93b1 introns (lines) and exons (blocks), including the mutant (dashed red line) splicing pattern and resulting mRNA transcripts for Unc93b1 +/+ and Unc93b1 Letr/Letr mice. (D) Multiple species alignment of the UNC93B1 amino acid sequence surrounding and including exon 4. The boxed area delineates the missing sequence in Unc93b1 Letr/Letr mice. (E) Predicted three-dimensional structure of wild type UNC93B1 with the 3d (red) and Letr (yellow) mutations mapped to their respective protein domains. The amino- and carboxy-termini are indicated as N and C, respectively.
Figure Legend Snippet: The Letr allele causes a single nucleotide transversion and alternative splicing of Unc93b1 (A) Chromatogram of the Unc93b1 +/+ (top) and Unc93b1 Letr/Letr (bottom) gDNA sequence with the single T to A nucleotide change caused by the Letr mutation that is identified on the non-coding strand as A to T. (B) Gel electrophoresis of cDNA from Unc93b1 Letr/Letr , Unc93b1 +/Letr , and wild type inbred strains (C57BL/6, 129S1, C3H/HeN). The wild type transcript size is 918bp while the mutant transcript size is 756bp. (C) Schematic of Unc93b1 introns (lines) and exons (blocks), including the mutant (dashed red line) splicing pattern and resulting mRNA transcripts for Unc93b1 +/+ and Unc93b1 Letr/Letr mice. (D) Multiple species alignment of the UNC93B1 amino acid sequence surrounding and including exon 4. The boxed area delineates the missing sequence in Unc93b1 Letr/Letr mice. (E) Predicted three-dimensional structure of wild type UNC93B1 with the 3d (red) and Letr (yellow) mutations mapped to their respective protein domains. The amino- and carboxy-termini are indicated as N and C, respectively.

Techniques Used: Sequencing, Mutagenesis, Nucleic Acid Electrophoresis, Mouse Assay

34) Product Images from "An ENU-induced splicing mutation reveals a role for Unc93b1 in early immune cell activation following Influenza A H1N1 infection"

Article Title: An ENU-induced splicing mutation reveals a role for Unc93b1 in early immune cell activation following Influenza A H1N1 infection

Journal: Genes and immunity

doi: 10.1038/gene.2014.22

The Letr allele causes a single nucleotide transversion and alternative splicing of Unc93b1 (A) Chromatogram of the Unc93b1 +/+ (top) and Unc93b1 Letr/Letr (bottom) gDNA sequence with the single T to A nucleotide change caused by the Letr mutation that is identified on the non-coding strand as A to T. (B) Gel electrophoresis of cDNA from Unc93b1 Letr/Letr , Unc93b1 +/Letr , and wild type inbred strains (C57BL/6, 129S1, C3H/HeN). The wild type transcript size is 918bp while the mutant transcript size is 756bp. (C) Schematic of Unc93b1 introns (lines) and exons (blocks), including the mutant (dashed red line) splicing pattern and resulting mRNA transcripts for Unc93b1 +/+ and Unc93b1 Letr/Letr mice. (D) Multiple species alignment of the UNC93B1 amino acid sequence surrounding and including exon 4. The boxed area delineates the missing sequence in Unc93b1 Letr/Letr mice. (E) Predicted three-dimensional structure of wild type UNC93B1 with the 3d (red) and Letr (yellow) mutations mapped to their respective protein domains. The amino- and carboxy-termini are indicated as N and C, respectively.
Figure Legend Snippet: The Letr allele causes a single nucleotide transversion and alternative splicing of Unc93b1 (A) Chromatogram of the Unc93b1 +/+ (top) and Unc93b1 Letr/Letr (bottom) gDNA sequence with the single T to A nucleotide change caused by the Letr mutation that is identified on the non-coding strand as A to T. (B) Gel electrophoresis of cDNA from Unc93b1 Letr/Letr , Unc93b1 +/Letr , and wild type inbred strains (C57BL/6, 129S1, C3H/HeN). The wild type transcript size is 918bp while the mutant transcript size is 756bp. (C) Schematic of Unc93b1 introns (lines) and exons (blocks), including the mutant (dashed red line) splicing pattern and resulting mRNA transcripts for Unc93b1 +/+ and Unc93b1 Letr/Letr mice. (D) Multiple species alignment of the UNC93B1 amino acid sequence surrounding and including exon 4. The boxed area delineates the missing sequence in Unc93b1 Letr/Letr mice. (E) Predicted three-dimensional structure of wild type UNC93B1 with the 3d (red) and Letr (yellow) mutations mapped to their respective protein domains. The amino- and carboxy-termini are indicated as N and C, respectively.

Techniques Used: Sequencing, Mutagenesis, Nucleic Acid Electrophoresis, Mouse Assay

35) Product Images from "5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic biomarkers for human cancers"

Article Title: 5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic biomarkers for human cancers

Journal: Cell Research

doi: 10.1038/cr.2017.121

Performance of 5hmC biomarkers for gastric cancer. (A) The heat map shows clustering of cfDNA samples from both the discovery and validation batches, using the 1 431 differential gene bodies detected in plasma cfDNA from the discovery batch. (B) Correlation of 5hmC changes in cancer between the discovery and validation batches is higher in plasma cfDNA (cancer patients vs healthy individuals) than in tumor gDNA (tumors vs adjacent tissues), especially for 5hmC in gene bodies. (C , D) Classifying two independent validation batches using 5hmC classifier derived from plasma cfDNA from the discovery batch. (E) Classifying an independent set of gastric cancer tumor tissues using 5hmC biomarkers detected from the discovery batch of tissue samples (tumors vs adjacent tissues). (F) The predicted cancer probability (i.e., score) based on the 5hmC classifier from plasma cfDNA shows a trend associated with clinical stage. The one patient after surgery shows a predicted probability undistinguishable from healthy individuals. (G) The 5hmC cfDNA classifier for gastric cancer is disease- and potentially cancer type-specific, showing decreasing predicted probability in cfDNA from colorectal, liver, pancreatic and thyroid cancer patients.
Figure Legend Snippet: Performance of 5hmC biomarkers for gastric cancer. (A) The heat map shows clustering of cfDNA samples from both the discovery and validation batches, using the 1 431 differential gene bodies detected in plasma cfDNA from the discovery batch. (B) Correlation of 5hmC changes in cancer between the discovery and validation batches is higher in plasma cfDNA (cancer patients vs healthy individuals) than in tumor gDNA (tumors vs adjacent tissues), especially for 5hmC in gene bodies. (C , D) Classifying two independent validation batches using 5hmC classifier derived from plasma cfDNA from the discovery batch. (E) Classifying an independent set of gastric cancer tumor tissues using 5hmC biomarkers detected from the discovery batch of tissue samples (tumors vs adjacent tissues). (F) The predicted cancer probability (i.e., score) based on the 5hmC classifier from plasma cfDNA shows a trend associated with clinical stage. The one patient after surgery shows a predicted probability undistinguishable from healthy individuals. (G) The 5hmC cfDNA classifier for gastric cancer is disease- and potentially cancer type-specific, showing decreasing predicted probability in cfDNA from colorectal, liver, pancreatic and thyroid cancer patients.

Techniques Used: Derivative Assay

Differential 5hmC loci associated with cancer. (A) Average 5hmC levels in gene bodies in healthy controls (health) and cancer patients (colon, stomach), estimated for plasma cfDNA (plasma cf), white blood cell genomic DNA (WBC) and tissue genomic DNA (tumor, adjacent), were clustered by correlation distance. (B) Counts per million reads at SULF1 gene (plus ± 20 kb region) in plasma cfDNA of the 15 healthy controls and 18 colorectal cancer patients. The moving averages at 0.01 smoother span are shown. (C) The distribution of colorectal cancer-associated 5hmC loci detected at 5% false discovery rate in plasma cfDNA. Each vertical bar denotes a differential locus (a histone modification peak or a gene body). The color key indicates the relative magnitude of log 2 fold change in cancer patients vs controls. (D) Pearson's correlation of log 2 fold changes between all analyzed genes and their neighboring genes (points) was plotted against the null distribution of correlation with their first neighboring genes (curves), generated by shuffling gene positions for 1 000 times. Blue and orange points denote data from plasma cfDNA and tissue gDNA, respectively, for colorectal cancer. In C and D , chromosome 1 is shown as an example. (E) Cancer plasma cfDNA and tumor gDNA exhibit correlation in average 5hmC density (library size and feature length normalized log 2 counts, black bars). However, there is no correlation in the log 2 fold changes between differential 5hmC loci detected (between cancer vs health (plasma cfDNA)) and tumor vs adjacent tissue (tissue gDNA), (orange bars). (F) Genes with a 5hmC level elevated in cancer plasma cfDNA (cancer cf) are enriched in genes with high 5hmC level in tissue gDNA (tumor high, adjacent high). To estimate fold enrichment, the 1st, 5th and 10th percentile genes in descending order of the log 2 fold change in cancer cfDNA were compared against the corresponding percentile genes in descending order of the average 5hmC level in tissue gDNA. Similarly, genes with a 5hmC level decreased in cancer plasma, and cfDNA are enriched in genes with low 5hmC levels in tissue gDNA (tumor low, adjacent low). In contrast, differentially marked genes detected in tumor gDNA (tumor) show no such enrichment pattern. Dashed line denotes no enrichment.
Figure Legend Snippet: Differential 5hmC loci associated with cancer. (A) Average 5hmC levels in gene bodies in healthy controls (health) and cancer patients (colon, stomach), estimated for plasma cfDNA (plasma cf), white blood cell genomic DNA (WBC) and tissue genomic DNA (tumor, adjacent), were clustered by correlation distance. (B) Counts per million reads at SULF1 gene (plus ± 20 kb region) in plasma cfDNA of the 15 healthy controls and 18 colorectal cancer patients. The moving averages at 0.01 smoother span are shown. (C) The distribution of colorectal cancer-associated 5hmC loci detected at 5% false discovery rate in plasma cfDNA. Each vertical bar denotes a differential locus (a histone modification peak or a gene body). The color key indicates the relative magnitude of log 2 fold change in cancer patients vs controls. (D) Pearson's correlation of log 2 fold changes between all analyzed genes and their neighboring genes (points) was plotted against the null distribution of correlation with their first neighboring genes (curves), generated by shuffling gene positions for 1 000 times. Blue and orange points denote data from plasma cfDNA and tissue gDNA, respectively, for colorectal cancer. In C and D , chromosome 1 is shown as an example. (E) Cancer plasma cfDNA and tumor gDNA exhibit correlation in average 5hmC density (library size and feature length normalized log 2 counts, black bars). However, there is no correlation in the log 2 fold changes between differential 5hmC loci detected (between cancer vs health (plasma cfDNA)) and tumor vs adjacent tissue (tissue gDNA), (orange bars). (F) Genes with a 5hmC level elevated in cancer plasma cfDNA (cancer cf) are enriched in genes with high 5hmC level in tissue gDNA (tumor high, adjacent high). To estimate fold enrichment, the 1st, 5th and 10th percentile genes in descending order of the log 2 fold change in cancer cfDNA were compared against the corresponding percentile genes in descending order of the average 5hmC level in tissue gDNA. Similarly, genes with a 5hmC level decreased in cancer plasma, and cfDNA are enriched in genes with low 5hmC levels in tissue gDNA (tumor low, adjacent low). In contrast, differentially marked genes detected in tumor gDNA (tumor) show no such enrichment pattern. Dashed line denotes no enrichment.

Techniques Used: Modification, Generated

Performance of 5hmC biomarkers for colorectal cancer. (A) The heat map shows clustering of cfDNA samples from both the discovery and validation batches, using the 989 differential gene bodies detected in plasma cfDNA from the discovery batch. (B) Correlation of 5hmC changes in cancer between the discovery and validation batches is higher in plasma cfDNA (cancer patients vs healthy individuals) than in tissue gDNA (tumors vs adjacent tissues), especially for 5hmC in gene bodies. (C , D) Classification of two independent validation batches using 5hmC classifier derived from plasma cfDNA from the discovery batch. (E) Classification of an independent set of colon cancer tumor tissues using 5hmC biomarkers detected from the discovery batch of tissue samples (tumors vs adjacent tissues). (F) The predicted cancer probability (i.e., score) based on 5hmC classifier from plasma cfDNA shows a significant trend associated with clinical stage. Patients after surgery show predicted scores undistinguishable from healthy individuals. (G) The 5hmC cfDNA classifier for colorectal cancer is disease- and potentially cancer type-specific, showing decreasing predicted probability in cfDNA from stomach, liver, pancreatic and thyroid cancer patients. AUC, area under curve; CAC, cancer patients; HEA, healthy controls; NOR, patients with benign tumor.
Figure Legend Snippet: Performance of 5hmC biomarkers for colorectal cancer. (A) The heat map shows clustering of cfDNA samples from both the discovery and validation batches, using the 989 differential gene bodies detected in plasma cfDNA from the discovery batch. (B) Correlation of 5hmC changes in cancer between the discovery and validation batches is higher in plasma cfDNA (cancer patients vs healthy individuals) than in tissue gDNA (tumors vs adjacent tissues), especially for 5hmC in gene bodies. (C , D) Classification of two independent validation batches using 5hmC classifier derived from plasma cfDNA from the discovery batch. (E) Classification of an independent set of colon cancer tumor tissues using 5hmC biomarkers detected from the discovery batch of tissue samples (tumors vs adjacent tissues). (F) The predicted cancer probability (i.e., score) based on 5hmC classifier from plasma cfDNA shows a significant trend associated with clinical stage. Patients after surgery show predicted scores undistinguishable from healthy individuals. (G) The 5hmC cfDNA classifier for colorectal cancer is disease- and potentially cancer type-specific, showing decreasing predicted probability in cfDNA from stomach, liver, pancreatic and thyroid cancer patients. AUC, area under curve; CAC, cancer patients; HEA, healthy controls; NOR, patients with benign tumor.

Techniques Used: Derivative Assay

The origin of cancer-associated 5hmC changes observed in plasma cfDNA. (A) The proportions of human reads in plasma cfDNA captured with 5hmC-Seal are shown for PDX mice grafted with tumor from three gastric cancer patients (stomach_1-3), three colorectal cancer patients (colon_1-3) and for PDX mice without graft (control_1-3). Vertical bars represent s.d. estimated from three replicate PDX mice for each patient. The PDX mice grafted with gastric tumor had greater number of passages (6-10) than those grafted with colorectal tumor (2-5). (B) The correlation of the 5hmC profile between tumor-derived, PDX plasma cfDNA and donor tumor gDNA depends on the number of passages of the PDX mouse. The size of the points is proportional to the size of grafted tumor, and the density of color denotes the growth rate of the grafted tumor. (C) Using the correlation distance of the top five genes that had the greatest 5hmC level in PDX plasma cfDNA, donor tumor gDNA and PDX plasma cfDNA from the same individual patient were clustered together. (D) Genes with elevated 5hmC level in cancer patient plasma cfDNA are enriched in genes with high 5hmC levels in PDX plasma cfDNA. To estimate fold enrichment, the fifth percentile genes in descending order of the log 2 fold change in patient cfDNA were compared against the corresponding fifth percentile genes in descending order of the 5hmC level in PDX mice (stomach pdx high, colon pdx high). Similarly, genes with decreased 5hmC level in cancer patient plasma cfDNA are enriched in genes with low 5hmC levels in PDX plasma cfDNA (stomach pdx low, colon pdx low). Dashed line denotes no enrichment.
Figure Legend Snippet: The origin of cancer-associated 5hmC changes observed in plasma cfDNA. (A) The proportions of human reads in plasma cfDNA captured with 5hmC-Seal are shown for PDX mice grafted with tumor from three gastric cancer patients (stomach_1-3), three colorectal cancer patients (colon_1-3) and for PDX mice without graft (control_1-3). Vertical bars represent s.d. estimated from three replicate PDX mice for each patient. The PDX mice grafted with gastric tumor had greater number of passages (6-10) than those grafted with colorectal tumor (2-5). (B) The correlation of the 5hmC profile between tumor-derived, PDX plasma cfDNA and donor tumor gDNA depends on the number of passages of the PDX mouse. The size of the points is proportional to the size of grafted tumor, and the density of color denotes the growth rate of the grafted tumor. (C) Using the correlation distance of the top five genes that had the greatest 5hmC level in PDX plasma cfDNA, donor tumor gDNA and PDX plasma cfDNA from the same individual patient were clustered together. (D) Genes with elevated 5hmC level in cancer patient plasma cfDNA are enriched in genes with high 5hmC levels in PDX plasma cfDNA. To estimate fold enrichment, the fifth percentile genes in descending order of the log 2 fold change in patient cfDNA were compared against the corresponding fifth percentile genes in descending order of the 5hmC level in PDX mice (stomach pdx high, colon pdx high). Similarly, genes with decreased 5hmC level in cancer patient plasma cfDNA are enriched in genes with low 5hmC levels in PDX plasma cfDNA (stomach pdx low, colon pdx low). Dashed line denotes no enrichment.

Techniques Used: Mouse Assay, Derivative Assay

36) Product Images from "Overview of methodologies for T-cell receptor repertoire analysis"

Article Title: Overview of methodologies for T-cell receptor repertoire analysis

Journal: BMC Biotechnology

doi: 10.1186/s12896-017-0379-9

Exemplary workflow of three principal methodologies for TCR library preparation. The figure depicts a simplified workflow of the library preparation procedure using multiplex PCR, targeted in-solution enrichment and 5’RACE-switch-oligo nested PCR. Multiplex PCR is suitable for both RNA and gDNA sequencing. Samples undergo cDNA synthesis and 1 or more PCR steps followed by adaptor ligation and sequencing. While the forward primers for cDNA synthesis are designed to cover all known V genes for both starting materials, the location and number of the reverse primers differs, due to introns in DNA. Target enrichment, also applicable to both gDNA and RNA, is preceded by a standard library preparation including fragmentation for gDNA or mRNA purification for RNA, followed by end-repairing, A-tailing and finally adaptor ligation. The enrichment of target sequences is then performed using RNA baits complementary to the sequence of interest. The RNA baits hybridize with molecules in the library, which are then retrieved using magnetic beads and can undergo further amplification before sequencing. Nested PCR based on the 5’RACE and switch-oligo approach (only for RNA) makes use of the incorporation of an adaptor molecule at the 5′ end of the cDNA during cDNA synthesis. The forward primer for a subsequent PCR is designed to bind to the 5′ adaptor sequence, while the reverse primer is designed to bind to the C-region of the transcript. Hence, only one primer pair is required to cover the complete spectrum of possible V genes. Subsequent nested PCRs performed in the same fashion may increase outcome specificity. Finally, adaptor ligation is performed. The procedures showed in this picture constitute only an example of the different available methods
Figure Legend Snippet: Exemplary workflow of three principal methodologies for TCR library preparation. The figure depicts a simplified workflow of the library preparation procedure using multiplex PCR, targeted in-solution enrichment and 5’RACE-switch-oligo nested PCR. Multiplex PCR is suitable for both RNA and gDNA sequencing. Samples undergo cDNA synthesis and 1 or more PCR steps followed by adaptor ligation and sequencing. While the forward primers for cDNA synthesis are designed to cover all known V genes for both starting materials, the location and number of the reverse primers differs, due to introns in DNA. Target enrichment, also applicable to both gDNA and RNA, is preceded by a standard library preparation including fragmentation for gDNA or mRNA purification for RNA, followed by end-repairing, A-tailing and finally adaptor ligation. The enrichment of target sequences is then performed using RNA baits complementary to the sequence of interest. The RNA baits hybridize with molecules in the library, which are then retrieved using magnetic beads and can undergo further amplification before sequencing. Nested PCR based on the 5’RACE and switch-oligo approach (only for RNA) makes use of the incorporation of an adaptor molecule at the 5′ end of the cDNA during cDNA synthesis. The forward primer for a subsequent PCR is designed to bind to the 5′ adaptor sequence, while the reverse primer is designed to bind to the C-region of the transcript. Hence, only one primer pair is required to cover the complete spectrum of possible V genes. Subsequent nested PCRs performed in the same fashion may increase outcome specificity. Finally, adaptor ligation is performed. The procedures showed in this picture constitute only an example of the different available methods

Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Nested PCR, Sequencing, Ligation, Purification, Magnetic Beads, Amplification

37) Product Images from "Cytoplasmic body pathology in severe ACTA1-related myopathy in the absence of typical nemaline rods"

Article Title: Cytoplasmic body pathology in severe ACTA1-related myopathy in the absence of typical nemaline rods

Journal: Neuromuscular disorders : NMD

doi: 10.1016/j.nmd.2017.02.012

Pedigree of Family 2 (A) and sequence chromatograms of targeted Sanger sequencing validation of the ACTA1 mutation identified on exome (B). Mutations were confirmed on gDNA extracted from blood. Sequencing results of Family 2 showing heterozygous c.282C > A (p.Asn94Lys) ACTA1 mutation in patients P2 and P3 (bottom), which was not identified in the father or mother (top).
Figure Legend Snippet: Pedigree of Family 2 (A) and sequence chromatograms of targeted Sanger sequencing validation of the ACTA1 mutation identified on exome (B). Mutations were confirmed on gDNA extracted from blood. Sequencing results of Family 2 showing heterozygous c.282C > A (p.Asn94Lys) ACTA1 mutation in patients P2 and P3 (bottom), which was not identified in the father or mother (top).

Techniques Used: Sequencing, Mutagenesis

38) Product Images from "Sequence and expression analysis of gaps in human chromosome 20"

Article Title: Sequence and expression analysis of gaps in human chromosome 20

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks302

Chromatin distribution and DNA methylation profile across human chr 20-gap regions. ( A ) H3K27me3 is distributed across all three human chr-20 gap regions in EBV-PBLs. ( B ) MRE-Seq (hypomethylated) and MBD-Seq (hypermethylated) aligned peak reads within gaps 1 and 3. Methylation distribution is similar in PBL and human cerebellum for both gaps 1 and 3. ( C ) Methylation profile of gap 2 shows it to be enriched for both hypomethylated and hypermethylated CpGs, respectively. CpG island 4/1a (red rectangle) is enriched for both MBD-Seq and MRE-Seq reads suggesting it to be a differentially methylated locus. CpG islands 2–6 were annotated using the classification: 500-bp region of genomic DNA with ≥50% CG content and an observed CpG to expected CpG ratio of 0.6. CpG islands 1a–3a were classified based on a CpG island being a 500-bp region of genomic DNA with ≥55% CG content and an observed CpG to expected CpG ratio of 0.65. Images are drawn to scale. ( D ) Differentially methylated and paternally hypermethylated CpGs island 4/1a within gap 2 using bisulfite allelic sequencing and gDNA from mouse–human cell hybrid cell lines or diploid human cells.
Figure Legend Snippet: Chromatin distribution and DNA methylation profile across human chr 20-gap regions. ( A ) H3K27me3 is distributed across all three human chr-20 gap regions in EBV-PBLs. ( B ) MRE-Seq (hypomethylated) and MBD-Seq (hypermethylated) aligned peak reads within gaps 1 and 3. Methylation distribution is similar in PBL and human cerebellum for both gaps 1 and 3. ( C ) Methylation profile of gap 2 shows it to be enriched for both hypomethylated and hypermethylated CpGs, respectively. CpG island 4/1a (red rectangle) is enriched for both MBD-Seq and MRE-Seq reads suggesting it to be a differentially methylated locus. CpG islands 2–6 were annotated using the classification: 500-bp region of genomic DNA with ≥50% CG content and an observed CpG to expected CpG ratio of 0.6. CpG islands 1a–3a were classified based on a CpG island being a 500-bp region of genomic DNA with ≥55% CG content and an observed CpG to expected CpG ratio of 0.65. Images are drawn to scale. ( D ) Differentially methylated and paternally hypermethylated CpGs island 4/1a within gap 2 using bisulfite allelic sequencing and gDNA from mouse–human cell hybrid cell lines or diploid human cells.

Techniques Used: DNA Methylation Assay, Methylation, Sequencing

39) Product Images from "Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing"

Article Title: Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing

Journal: Plant Methods

doi: 10.1186/s13007-018-0336-1

Influence of genomic DNA (gDNA) extraction protocols on sequencing quality. We assessed and modified methodologies to establish a high-throughput protocol to extract gDNA from white clover, a species prone to yielding degraded DNA. Chemistry based on a CTAB protocol [ 6 ] was modified for gDNA extraction using 96-well plates ( a ). Adaptation of the Whitlock method [ 2 ] to a 96-well plate protocol ( b ), and development of a streamlined inexpensive protocol described in this paper ( c ). In these examples, gDNA was extracted from freeze-dried leaves and aliquots (2 μL from 100 μL gDNA extraction/elution) were resolved and visualised by electrophoresis in an agarose lithium borate buffer (0.8% w/v) gel containing 25 μg ethidium bromide. The samples were flanked at either end by 1 kb Plus size ladders ( www.thermofisher.com ). Sequence quality of the extracted gDNA was assessed by producing a genotyping-by-sequencing (GBS) library [ 1 ] comprising 95 individuals from each of the white clover gDNA extractions shown above ( a – c ). The GBS libraries were single-end sequenced (100 bp) on a single lane each of an Illumina 2500 Hi-Seq sequencer. Sequencing quality assessment using FastQC version 0.10.1 [ 9 ] for GBS libraries made from the gDNA shown a – c is represented in graphs describing quality across all bases from every sequence read at each position ( d – f , respectively). Sequence quality is based on phred scores [ 10 ], an exponential scale where, for example, 20 = one incorrect sequence base-call in 100, and 30 = one incorrect base-call in 1000. The y-axis shows the quality scores, and the higher the score the greater confidence in the base-calls at that position. The background of the graph divides the y-axis into very good quality calls (green), reasonable quality (orange), and poor quality (red)
Figure Legend Snippet: Influence of genomic DNA (gDNA) extraction protocols on sequencing quality. We assessed and modified methodologies to establish a high-throughput protocol to extract gDNA from white clover, a species prone to yielding degraded DNA. Chemistry based on a CTAB protocol [ 6 ] was modified for gDNA extraction using 96-well plates ( a ). Adaptation of the Whitlock method [ 2 ] to a 96-well plate protocol ( b ), and development of a streamlined inexpensive protocol described in this paper ( c ). In these examples, gDNA was extracted from freeze-dried leaves and aliquots (2 μL from 100 μL gDNA extraction/elution) were resolved and visualised by electrophoresis in an agarose lithium borate buffer (0.8% w/v) gel containing 25 μg ethidium bromide. The samples were flanked at either end by 1 kb Plus size ladders ( www.thermofisher.com ). Sequence quality of the extracted gDNA was assessed by producing a genotyping-by-sequencing (GBS) library [ 1 ] comprising 95 individuals from each of the white clover gDNA extractions shown above ( a – c ). The GBS libraries were single-end sequenced (100 bp) on a single lane each of an Illumina 2500 Hi-Seq sequencer. Sequencing quality assessment using FastQC version 0.10.1 [ 9 ] for GBS libraries made from the gDNA shown a – c is represented in graphs describing quality across all bases from every sequence read at each position ( d – f , respectively). Sequence quality is based on phred scores [ 10 ], an exponential scale where, for example, 20 = one incorrect sequence base-call in 100, and 30 = one incorrect base-call in 1000. The y-axis shows the quality scores, and the higher the score the greater confidence in the base-calls at that position. The background of the graph divides the y-axis into very good quality calls (green), reasonable quality (orange), and poor quality (red)

Techniques Used: Sequencing, Modification, High Throughput Screening Assay, Electrophoresis

A typical DNA extraction from freeze-dried and fresh tissue using the 96-well plate method. Genomic DNA was extracted from 96 individuals of a Trifolium repens (white clover; freeze-dried leaf) and b Lolium perenne (perennial ryegrass; fresh pseudostem) using the 96-well plate protocols, and resolved and visualised by electrophoresis on an agarose lithium borate buffer (0.8% w/v) gel containing 25 μg ethidium bromide. The samples were flanked at either end by 1 Kb Plus size ladders ( www.thermofisher.com ). One T. repens individual had a poor DNA yield (top row, ninth lane from the right) and was subsequently re-extracted using the Individual Tube freeze-dried tissue protocol. These DNA samples have been used for developing genotyping-by-sequencing libraries and yielded high-quality sequence data
Figure Legend Snippet: A typical DNA extraction from freeze-dried and fresh tissue using the 96-well plate method. Genomic DNA was extracted from 96 individuals of a Trifolium repens (white clover; freeze-dried leaf) and b Lolium perenne (perennial ryegrass; fresh pseudostem) using the 96-well plate protocols, and resolved and visualised by electrophoresis on an agarose lithium borate buffer (0.8% w/v) gel containing 25 μg ethidium bromide. The samples were flanked at either end by 1 Kb Plus size ladders ( www.thermofisher.com ). One T. repens individual had a poor DNA yield (top row, ninth lane from the right) and was subsequently re-extracted using the Individual Tube freeze-dried tissue protocol. These DNA samples have been used for developing genotyping-by-sequencing libraries and yielded high-quality sequence data

Techniques Used: DNA Extraction, Electrophoresis, Sequencing

40) Product Images from "Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma, et al. Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma"

Article Title: Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma, et al. Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma

Journal: Cancer Science

doi: 10.1111/cas.13906

Somatic mutations detected by targeted sequencing of cell‐free DNA (cfDNA) and genomic DNA (gDNA) from tumor tissue. Mutated genes detected by targeted sequencing are shown in the left‐most column (arranged in descending order of the number of mutations). Numbers for each gene indicate the frequency of the mutant allele (%). MISSSENSE, missense mutation; NONSENSE, nonsense mutation; INDEL, insertion/deletion; SPLICING, splicing site mutation. A, Somatic mutations in plasma cfDNA and gDNA from cancer (n = 5). B, Somatic mutations of plasma cfDNA in 16 RCC patients with at least one mutation. Patient state at blood collection was classified as “Pretreatment without metastasis” (purple), “Pretreatment with metastasis” (green) and “Post‐treatment with metastasis or recurrence” (red). Right bar plot shows the number of samples
Figure Legend Snippet: Somatic mutations detected by targeted sequencing of cell‐free DNA (cfDNA) and genomic DNA (gDNA) from tumor tissue. Mutated genes detected by targeted sequencing are shown in the left‐most column (arranged in descending order of the number of mutations). Numbers for each gene indicate the frequency of the mutant allele (%). MISSSENSE, missense mutation; NONSENSE, nonsense mutation; INDEL, insertion/deletion; SPLICING, splicing site mutation. A, Somatic mutations in plasma cfDNA and gDNA from cancer (n = 5). B, Somatic mutations of plasma cfDNA in 16 RCC patients with at least one mutation. Patient state at blood collection was classified as “Pretreatment without metastasis” (purple), “Pretreatment with metastasis” (green) and “Post‐treatment with metastasis or recurrence” (red). Right bar plot shows the number of samples

Techniques Used: Sequencing, Mutagenesis

Related Articles

Amplification:

Article Title: HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression
Article Snippet: .. To define the amplification at the HPV16 integration site in 20861 cells, 2 x 150 bp paired-end libraries were generated from total genomic DNA and subjected to WGS using the HiSeq 4000 platform (Illumina Genome Network). .. WGS data were aligned to the human reference genome (hg19) with ~7X sequencing coverage ( ).

Methylation:

Article Title: Genome‐wide DNA methylation profiling shows molecular heterogeneity of anaplastic pleomorphic xanthoastrocytoma, et al. Genome‐wide DNA methylation profiling shows molecular heterogeneity of anaplastic pleomorphic xanthoastrocytoma
Article Snippet: .. Genomic DNA from the anaplastic PXA (anaplastic PXA01), two PXA (PXA10 and PXA11) were analyzed using an Infinium Human Methylation 450K BeadChip (Illumina, San Diego, CA, USA) (GPL13534) as described previously (Data [Illumina Infinium 450K Methylation Array data generation]). .. DNA methylation and clinical data of nine pediatric PXA and 50 GBM generated by Sturm et al (GSE73801) were obtained from the GEO database for comparison (Table ).

DNA Methylation Assay:

Article Title: Hydroxylation of 5-methylcytosine by TET2 maintains the active state of the mammalian HOXA cluster
Article Snippet: .. In order to study detailed DNA methylation patterns before and after RA induction, we analysed genomic DNA of RA-treated and untreated NT2 cells using Infinium450K BeadChips (Illumina). .. These arrays interrogate more than 450,000 methylation sites in the human genome, including CpG islands (CGIs), CpG sites outside of CGIs and non-CpG methylation sites identified in human embryonic stem cells .

Sequencing:

Article Title: Choice of Reference Sequence and Assembler for Alignment of Listeria monocytogenes Short-Read Sequence Data Greatly Influences Rates of Error in SNP Analyses
Article Snippet: .. Results and Discussion We assessed the efficacy of four commonly used reference-guided short-read sequence assemblers (BWA, MOSAIK, Novoalign, and SMALT) to generate alignments suitable for accurate detection of single-nucleotide polymorphisms (SNPs) using both simulated reads and actual reads obtained from sequencing runs of Listeria monocytogenes strain 08-5578 genomic DNA on an Illumina MiSeq benchtop machine. ..

Article Title: The long non-coding RNA Paupar regulates the expression of both local and distal genes
Article Snippet: .. Following the CHART-seq protocol, we used RNase H elution to recover genomic DNA associated with endogenous Paupar transcripts and genomic DNA associated with the control oligonucleotide, sequencing replicate samples using the Illumina HiSeq system. .. Using the paired-end peak caller MACS2 (Zhang et al , ), we identified Paupar binding sites in comparison both to DNA recovered using the control LacZ oligonucleotide and to input DNA from N2A cells.

Article Title: Overview of methodologies for T-cell receptor repertoire analysis
Article Snippet: .. This kit is highly customizable and offers options for both T and B cell receptor sequencing, different receptor chains (αβ, γδ), gDNA or RNA, mouse or human, and sequencing platforms (Illumina, Roche 454). iRprofile contains separate reactions mixes for α and β chains, each uniquely barcoded. .. The protocol consists of two consecutive PCRs using multiplex primers specific for the V and J genes of the mentioned chains.

Article Title: Saccharomyces cerevisiae DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity
Article Snippet: .. Genomic DNA was collected from wild-type, dnl4 -K466A, and dnl4Δ strains at 0-hour and 24-hour time points and PCR products flanking the HO DSB site were subjected to Illumina HiSeq sequencing. ..

Generated:

Article Title: HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression
Article Snippet: .. To define the amplification at the HPV16 integration site in 20861 cells, 2 x 150 bp paired-end libraries were generated from total genomic DNA and subjected to WGS using the HiSeq 4000 platform (Illumina Genome Network). .. WGS data were aligned to the human reference genome (hg19) with ~7X sequencing coverage ( ).

Polymerase Chain Reaction:

Article Title: Saccharomyces cerevisiae DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity
Article Snippet: .. Genomic DNA was collected from wild-type, dnl4 -K466A, and dnl4Δ strains at 0-hour and 24-hour time points and PCR products flanking the HO DSB site were subjected to Illumina HiSeq sequencing. ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Illumina Inc ctab extracted gdna
    Influence of genomic DNA <t>(gDNA)</t> extraction protocols on sequencing quality. We assessed and modified methodologies to establish a high-throughput protocol to extract gDNA from white clover, a species prone to yielding degraded DNA. Chemistry based on a <t>CTAB</t> protocol [ 6 ] was modified for gDNA extraction using 96-well plates ( a ). Adaptation of the Whitlock method [ 2 ] to a 96-well plate protocol ( b ), and development of a streamlined inexpensive protocol described in this paper ( c ). In these examples, gDNA was extracted from freeze-dried leaves and aliquots (2 μL from 100 μL gDNA extraction/elution) were resolved and visualised by electrophoresis in an agarose lithium borate buffer (0.8% w/v) gel containing 25 μg ethidium bromide. The samples were flanked at either end by 1 kb Plus size ladders ( www.thermofisher.com ). Sequence quality of the extracted gDNA was assessed by producing a genotyping-by-sequencing (GBS) library [ 1 ] comprising 95 individuals from each of the white clover gDNA extractions shown above ( a – c ). The GBS libraries were single-end sequenced (100 bp) on a single lane each of an Illumina 2500 Hi-Seq sequencer. Sequencing quality assessment using FastQC version 0.10.1 [ 9 ] for GBS libraries made from the gDNA shown a – c is represented in graphs describing quality across all bases from every sequence read at each position ( d – f , respectively). Sequence quality is based on phred scores [ 10 ], an exponential scale where, for example, 20 = one incorrect sequence base-call in 100, and 30 = one incorrect base-call in 1000. The y-axis shows the quality scores, and the higher the score the greater confidence in the base-calls at that position. The background of the graph divides the y-axis into very good quality calls (green), reasonable quality (orange), and poor quality (red)
    Ctab Extracted Gdna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ctab extracted gdna/product/Illumina Inc
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    ctab extracted gdna - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    85
    Illumina Inc paired end genomic dna library preparation kit
    Enrichment of chloroplast <t>DNA</t> from karaka . The relative quantity of chloroplast DNA was determined in preparations of genomic DNA (gDNA), chloroplast-enriched DNA (cpDNA) and RCA amplified chloroplast-enriched cpDNA (RCA). Quantitative <t>PCR</t> amplification of the plastid encoded psbB gene was compared to amplification of nuclear 18S rRNA . The mean of four technical replicates ± SE has been reported as fold-differences relative to gDNA.
    Paired End Genomic Dna Library Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/paired end genomic dna library preparation kit/product/Illumina Inc
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    paired end genomic dna library preparation kit - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    89
    Illumina Inc afb62f9 genomic dna
    Azole resistance experimental design. (A) Sensitive AFB62 or <t>AFB62F9</t> cells were incubated on solid media containing sub-MIC concentrations of each of itraconazole, voriconazole, or posaconazole. Resulting spores were plated on selective media with at least 1X MIC of each antifungal to obtain fewer than 100 colonies. Three resistant colonies (purple) from each selective plate were subsequently and independently plated on highly selective plates containing 10 – 50X MIC of each antifungal. Three highly resistant strains (purple) were selected for sequencing and for sexual crosses. (B) Each of the highly resistant isolates from (A) was crossed with the sensitive isogenic strain of the opposite mating type. Ascospores from selected crosses were grown on media with no drug, 1X MIC and 10X MIC of the drug and their <t>DNA</t> sequenced to identify resistance allele frequencies. Mutant alleles are depicted in purple and yellow reference alleles in green. Cells filled with a single color represent an allele frequency of 1, cells with two colors represent an equal proportion of mutant and reference alleles. The purple mutation shows the expected frequencies for an allele associated with resistance, while the yellow mutation shows an expected frequency for alleles not associated with resistance.
    Afb62f9 Genomic Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/afb62f9 genomic dna/product/Illumina Inc
    Average 89 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    afb62f9 genomic dna - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    90
    Illumina Inc chip enriched genomic dna
    Pc positively regulates Sens expression by regulating <t>H4K20me1</t> catalyzed by PR-Set7. (A - A''') A wing disc carrying PR-Set7 EY04668 mutant clones and immunostained with anti-Sens antibody (red), anti-CD2 (green) and anti-H4K20me1 antibody (blue). The clones are CD2 negative, and their boundary is demarcated with dashed lines. (B - C) RT-qPCR analysis of mRNA levels of PR-Set7 (B) and Sens (C) in Act5c-Gal4 or Act5c-Gal4/uas-dsRNA PR-Set7 wing discs. (D) ChIP-qPCR analysis using control IgG or anti-H4K20me1 antibody at the Sens locus with Act5c-Gal4 wing discs or Act5c-Gal4/uas-dsRNA PR-Set7 wing discs. Upper panel illustrates the positions of the primers used for the qPCR assay at the Sens locus. In the lower panel, ChIP signal levels are represented as percentages of input chromatin. (E) ChIP-qPCR analysis using control IgG or anti-H4K20me1 antibody with Act5c-Gal4 wing discs or Act5c-Gal4/uas-dsRNA Pc wing discs at the Sens locus. ChIP signal levels are represented as percentages of input chromatin. (F) S2 cells were transfected with combinations of <t>DNA</t> constructs as indicated. After 48 h of transfection, lysates from transfected S2 cells were immunoprecipitated with anti-Flag antibody or anti-Myc antibody. Western blots were performed to analyze the presence of Flag- or Myc-tagged proteins. (G - H) Mapping of individual domains responsible for the interaction of PR-Set7 with Pc. Schematic drawings of PR-Set7 full length (FL) protein and different fragments (G) . The SET domain that catalyzes H4K20me1 is located in the C-terminal (C) part of PR-Set7, while no significant domain is in the N-terminal (N) and middle (M) regions. A co-IP-WB shows that PR-Set7 interacts with Pc mainly through its M region (H) . Means ± SD; n = 3. n.s., no significant difference. * P
    Chip Enriched Genomic Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chip enriched genomic dna/product/Illumina Inc
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    chip enriched genomic dna - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    Image Search Results


    Influence of genomic DNA (gDNA) extraction protocols on sequencing quality. We assessed and modified methodologies to establish a high-throughput protocol to extract gDNA from white clover, a species prone to yielding degraded DNA. Chemistry based on a CTAB protocol [ 6 ] was modified for gDNA extraction using 96-well plates ( a ). Adaptation of the Whitlock method [ 2 ] to a 96-well plate protocol ( b ), and development of a streamlined inexpensive protocol described in this paper ( c ). In these examples, gDNA was extracted from freeze-dried leaves and aliquots (2 μL from 100 μL gDNA extraction/elution) were resolved and visualised by electrophoresis in an agarose lithium borate buffer (0.8% w/v) gel containing 25 μg ethidium bromide. The samples were flanked at either end by 1 kb Plus size ladders ( www.thermofisher.com ). Sequence quality of the extracted gDNA was assessed by producing a genotyping-by-sequencing (GBS) library [ 1 ] comprising 95 individuals from each of the white clover gDNA extractions shown above ( a – c ). The GBS libraries were single-end sequenced (100 bp) on a single lane each of an Illumina 2500 Hi-Seq sequencer. Sequencing quality assessment using FastQC version 0.10.1 [ 9 ] for GBS libraries made from the gDNA shown a – c is represented in graphs describing quality across all bases from every sequence read at each position ( d – f , respectively). Sequence quality is based on phred scores [ 10 ], an exponential scale where, for example, 20 = one incorrect sequence base-call in 100, and 30 = one incorrect base-call in 1000. The y-axis shows the quality scores, and the higher the score the greater confidence in the base-calls at that position. The background of the graph divides the y-axis into very good quality calls (green), reasonable quality (orange), and poor quality (red)

    Journal: Plant Methods

    Article Title: Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing

    doi: 10.1186/s13007-018-0336-1

    Figure Lengend Snippet: Influence of genomic DNA (gDNA) extraction protocols on sequencing quality. We assessed and modified methodologies to establish a high-throughput protocol to extract gDNA from white clover, a species prone to yielding degraded DNA. Chemistry based on a CTAB protocol [ 6 ] was modified for gDNA extraction using 96-well plates ( a ). Adaptation of the Whitlock method [ 2 ] to a 96-well plate protocol ( b ), and development of a streamlined inexpensive protocol described in this paper ( c ). In these examples, gDNA was extracted from freeze-dried leaves and aliquots (2 μL from 100 μL gDNA extraction/elution) were resolved and visualised by electrophoresis in an agarose lithium borate buffer (0.8% w/v) gel containing 25 μg ethidium bromide. The samples were flanked at either end by 1 kb Plus size ladders ( www.thermofisher.com ). Sequence quality of the extracted gDNA was assessed by producing a genotyping-by-sequencing (GBS) library [ 1 ] comprising 95 individuals from each of the white clover gDNA extractions shown above ( a – c ). The GBS libraries were single-end sequenced (100 bp) on a single lane each of an Illumina 2500 Hi-Seq sequencer. Sequencing quality assessment using FastQC version 0.10.1 [ 9 ] for GBS libraries made from the gDNA shown a – c is represented in graphs describing quality across all bases from every sequence read at each position ( d – f , respectively). Sequence quality is based on phred scores [ 10 ], an exponential scale where, for example, 20 = one incorrect sequence base-call in 100, and 30 = one incorrect base-call in 1000. The y-axis shows the quality scores, and the higher the score the greater confidence in the base-calls at that position. The background of the graph divides the y-axis into very good quality calls (green), reasonable quality (orange), and poor quality (red)

    Article Snippet: To assess sequencing quality of the gDNA, a genotyping-by-sequencing (GBS) library [ ] made from 95 white clover individuals using the CTAB-extracted gDNA shown (Fig. a) was single-end sequenced (100 bp) on a single lane of an Illumina HiSeq 2500 sequencer.

    Techniques: Sequencing, Modification, High Throughput Screening Assay, Electrophoresis

    Enrichment of chloroplast DNA from karaka . The relative quantity of chloroplast DNA was determined in preparations of genomic DNA (gDNA), chloroplast-enriched DNA (cpDNA) and RCA amplified chloroplast-enriched cpDNA (RCA). Quantitative PCR amplification of the plastid encoded psbB gene was compared to amplification of nuclear 18S rRNA . The mean of four technical replicates ± SE has been reported as fold-differences relative to gDNA.

    Journal: Plant Methods

    Article Title: Whole genome sequencing of enriched chloroplast DNA using the Illumina GAII platform

    doi: 10.1186/1746-4811-6-22

    Figure Lengend Snippet: Enrichment of chloroplast DNA from karaka . The relative quantity of chloroplast DNA was determined in preparations of genomic DNA (gDNA), chloroplast-enriched DNA (cpDNA) and RCA amplified chloroplast-enriched cpDNA (RCA). Quantitative PCR amplification of the plastid encoded psbB gene was compared to amplification of nuclear 18S rRNA . The mean of four technical replicates ± SE has been reported as fold-differences relative to gDNA.

    Article Snippet: Samples were prepared for sequencing as follows: genomic DNA libraries were prepared by fragmenting purified genomic DNA using a nebulisation kit (Invitrogen), paired-end index adaptor ligation (Illumina) and 18 cycles of PCR enrichment using the Illumina Paired-End Genomic DNA library preparation kit, Illumina Multiplex Oligonucleotide library preparation kit and Illumina Multiplex Paired-End Genomic DNA library preparation protocol.

    Techniques: Amplification, Real-time Polymerase Chain Reaction

    Azole resistance experimental design. (A) Sensitive AFB62 or AFB62F9 cells were incubated on solid media containing sub-MIC concentrations of each of itraconazole, voriconazole, or posaconazole. Resulting spores were plated on selective media with at least 1X MIC of each antifungal to obtain fewer than 100 colonies. Three resistant colonies (purple) from each selective plate were subsequently and independently plated on highly selective plates containing 10 – 50X MIC of each antifungal. Three highly resistant strains (purple) were selected for sequencing and for sexual crosses. (B) Each of the highly resistant isolates from (A) was crossed with the sensitive isogenic strain of the opposite mating type. Ascospores from selected crosses were grown on media with no drug, 1X MIC and 10X MIC of the drug and their DNA sequenced to identify resistance allele frequencies. Mutant alleles are depicted in purple and yellow reference alleles in green. Cells filled with a single color represent an allele frequency of 1, cells with two colors represent an equal proportion of mutant and reference alleles. The purple mutation shows the expected frequencies for an allele associated with resistance, while the yellow mutation shows an expected frequency for alleles not associated with resistance.

    Journal: PLoS Pathogens

    Article Title: Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus’s Role in Virulence

    doi: 10.1371/journal.ppat.1004834

    Figure Lengend Snippet: Azole resistance experimental design. (A) Sensitive AFB62 or AFB62F9 cells were incubated on solid media containing sub-MIC concentrations of each of itraconazole, voriconazole, or posaconazole. Resulting spores were plated on selective media with at least 1X MIC of each antifungal to obtain fewer than 100 colonies. Three resistant colonies (purple) from each selective plate were subsequently and independently plated on highly selective plates containing 10 – 50X MIC of each antifungal. Three highly resistant strains (purple) were selected for sequencing and for sexual crosses. (B) Each of the highly resistant isolates from (A) was crossed with the sensitive isogenic strain of the opposite mating type. Ascospores from selected crosses were grown on media with no drug, 1X MIC and 10X MIC of the drug and their DNA sequenced to identify resistance allele frequencies. Mutant alleles are depicted in purple and yellow reference alleles in green. Cells filled with a single color represent an allele frequency of 1, cells with two colors represent an equal proportion of mutant and reference alleles. The purple mutation shows the expected frequencies for an allele associated with resistance, while the yellow mutation shows an expected frequency for alleles not associated with resistance.

    Article Snippet: Genome sequencing of AFB62 and AFB62F9 Genomic DNA was sequenced using paired-end Illumina GA-II to 44-fold coverage.

    Techniques: Incubation, Sequencing, Mutagenesis

    Pc positively regulates Sens expression by regulating H4K20me1 catalyzed by PR-Set7. (A - A''') A wing disc carrying PR-Set7 EY04668 mutant clones and immunostained with anti-Sens antibody (red), anti-CD2 (green) and anti-H4K20me1 antibody (blue). The clones are CD2 negative, and their boundary is demarcated with dashed lines. (B - C) RT-qPCR analysis of mRNA levels of PR-Set7 (B) and Sens (C) in Act5c-Gal4 or Act5c-Gal4/uas-dsRNA PR-Set7 wing discs. (D) ChIP-qPCR analysis using control IgG or anti-H4K20me1 antibody at the Sens locus with Act5c-Gal4 wing discs or Act5c-Gal4/uas-dsRNA PR-Set7 wing discs. Upper panel illustrates the positions of the primers used for the qPCR assay at the Sens locus. In the lower panel, ChIP signal levels are represented as percentages of input chromatin. (E) ChIP-qPCR analysis using control IgG or anti-H4K20me1 antibody with Act5c-Gal4 wing discs or Act5c-Gal4/uas-dsRNA Pc wing discs at the Sens locus. ChIP signal levels are represented as percentages of input chromatin. (F) S2 cells were transfected with combinations of DNA constructs as indicated. After 48 h of transfection, lysates from transfected S2 cells were immunoprecipitated with anti-Flag antibody or anti-Myc antibody. Western blots were performed to analyze the presence of Flag- or Myc-tagged proteins. (G - H) Mapping of individual domains responsible for the interaction of PR-Set7 with Pc. Schematic drawings of PR-Set7 full length (FL) protein and different fragments (G) . The SET domain that catalyzes H4K20me1 is located in the C-terminal (C) part of PR-Set7, while no significant domain is in the N-terminal (N) and middle (M) regions. A co-IP-WB shows that PR-Set7 interacts with Pc mainly through its M region (H) . Means ± SD; n = 3. n.s., no significant difference. * P

    Journal: Cell Research

    Article Title: A positive role for polycomb in transcriptional regulation via H4K20me1

    doi: 10.1038/cr.2016.33

    Figure Lengend Snippet: Pc positively regulates Sens expression by regulating H4K20me1 catalyzed by PR-Set7. (A - A''') A wing disc carrying PR-Set7 EY04668 mutant clones and immunostained with anti-Sens antibody (red), anti-CD2 (green) and anti-H4K20me1 antibody (blue). The clones are CD2 negative, and their boundary is demarcated with dashed lines. (B - C) RT-qPCR analysis of mRNA levels of PR-Set7 (B) and Sens (C) in Act5c-Gal4 or Act5c-Gal4/uas-dsRNA PR-Set7 wing discs. (D) ChIP-qPCR analysis using control IgG or anti-H4K20me1 antibody at the Sens locus with Act5c-Gal4 wing discs or Act5c-Gal4/uas-dsRNA PR-Set7 wing discs. Upper panel illustrates the positions of the primers used for the qPCR assay at the Sens locus. In the lower panel, ChIP signal levels are represented as percentages of input chromatin. (E) ChIP-qPCR analysis using control IgG or anti-H4K20me1 antibody with Act5c-Gal4 wing discs or Act5c-Gal4/uas-dsRNA Pc wing discs at the Sens locus. ChIP signal levels are represented as percentages of input chromatin. (F) S2 cells were transfected with combinations of DNA constructs as indicated. After 48 h of transfection, lysates from transfected S2 cells were immunoprecipitated with anti-Flag antibody or anti-Myc antibody. Western blots were performed to analyze the presence of Flag- or Myc-tagged proteins. (G - H) Mapping of individual domains responsible for the interaction of PR-Set7 with Pc. Schematic drawings of PR-Set7 full length (FL) protein and different fragments (G) . The SET domain that catalyzes H4K20me1 is located in the C-terminal (C) part of PR-Set7, while no significant domain is in the N-terminal (N) and middle (M) regions. A co-IP-WB shows that PR-Set7 interacts with Pc mainly through its M region (H) . Means ± SD; n = 3. n.s., no significant difference. * P

    Article Snippet: Briefly, 10 ng ChIP-enriched genomic DNA with anti-H4K20me1 antibody was used for library preparation and sequenced from single-end on Illumina HiSeq 2000.

    Techniques: Expressing, Mutagenesis, Clone Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Construct, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay

    Selective markers for the functional switch of Pc from a transcription repressor to an activator. (A) Heat maps of ChIP signals at Pc + H3K27me3 + H4K20me1 − and Pc + H3K27me3 + H4K20me1 + genes. (B) The Broad/Forkhead motif identified in Pc + H3K27me3 + H4K20me1 + genes. (C) RT-qPCR analysis of mRNA levels of Br , Ry , Spn100A and Sens in Act5c-Gal4 or Act5c-Gal4/uas-dsRNA Br wing discs. (D) S2 cells were transfected with combinations of DNA constructs as indicated. After 48 h of transfection, lysates from transfected S2 cells were immunoprecipitated with anti-V5 antibody or anti-Myc antibody. Western blots were performed to analyze the presence of Flag-, V5- or Myc-tagged proteins. (E) Gel filtration chromatography on a Superose 6 column and western blot of Cl-8 nuclear extract. Molecular weight standards are shown on top (in kDa). Means ± SD; n = 3. * P

    Journal: Cell Research

    Article Title: A positive role for polycomb in transcriptional regulation via H4K20me1

    doi: 10.1038/cr.2016.33

    Figure Lengend Snippet: Selective markers for the functional switch of Pc from a transcription repressor to an activator. (A) Heat maps of ChIP signals at Pc + H3K27me3 + H4K20me1 − and Pc + H3K27me3 + H4K20me1 + genes. (B) The Broad/Forkhead motif identified in Pc + H3K27me3 + H4K20me1 + genes. (C) RT-qPCR analysis of mRNA levels of Br , Ry , Spn100A and Sens in Act5c-Gal4 or Act5c-Gal4/uas-dsRNA Br wing discs. (D) S2 cells were transfected with combinations of DNA constructs as indicated. After 48 h of transfection, lysates from transfected S2 cells were immunoprecipitated with anti-V5 antibody or anti-Myc antibody. Western blots were performed to analyze the presence of Flag-, V5- or Myc-tagged proteins. (E) Gel filtration chromatography on a Superose 6 column and western blot of Cl-8 nuclear extract. Molecular weight standards are shown on top (in kDa). Means ± SD; n = 3. * P

    Article Snippet: Briefly, 10 ng ChIP-enriched genomic DNA with anti-H4K20me1 antibody was used for library preparation and sequenced from single-end on Illumina HiSeq 2000.

    Techniques: Functional Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Transfection, Construct, Immunoprecipitation, Western Blot, Filtration, Chromatography, Molecular Weight