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Targeted sequencing of large genomic regions with CATCH-Seq.

Day K, Song J, Absher D - PLoS ONE (2014)

Bottom Line: Furthermore, libraries constructed with methylated adapters prior to solution hybridization also enable targeted bisulfite sequencing.We applied CATCH-Seq to diverse targets ranging in size from 125 kb to 3.5 Mb.Given its similarity in procedure, CATCH-Seq can also be performed in parallel with commercial systems.

View Article: PubMed Central - PubMed

Affiliation: HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, United States of America.

ABSTRACT
Current target enrichment systems for large-scale next-generation sequencing typically require synthetic oligonucleotides used as capture reagents to isolate sequences of interest. The majority of target enrichment reagents are focused on gene coding regions or promoters en masse. Here we introduce development of a customizable targeted capture system using biotinylated RNA probe baits transcribed from sheared bacterial artificial chromosome clone templates that enables capture of large, contiguous blocks of the genome for sequencing applications. This clone adapted template capture hybridization sequencing (CATCH-Seq) procedure can be used to capture both coding and non-coding regions of a gene, and resolve the boundaries of copy number variations within a genomic target site. Furthermore, libraries constructed with methylated adapters prior to solution hybridization also enable targeted bisulfite sequencing. We applied CATCH-Seq to diverse targets ranging in size from 125 kb to 3.5 Mb. Our approach provides a simple and cost effective alternative to other capture platforms because of template-based, enzymatic probe synthesis and the lack of oligonucleotide design costs. Given its similarity in procedure, CATCH-Seq can also be performed in parallel with commercial systems.

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High density methylation data derived from bisulfite sequencing of a CATCH-Seq target.Scale of the captured region is indicated in the topmost track in kilobases (kb), followed by repeat structure in gray and black (RepMask), genes shown in blue (RefSeq), and CpG islands in green. Four CATCH-Seq tracks from the same cell type show DNA methylation levels across ∼2,700 target CpGs with hypomethylation depicted in green and hypermethylation in red. Six reduced representation bisulfite sequencing (RRBS) tracks for different cell and tissue types correspond with the same captured region, and demonstrate CpGs not covered by RRBS method compared to CATCH-Seq. The four CATCH-Seq tracks are from the same cell type as the topmost RRBS track. RRBS tracks are derived from previously reported data [23]. CpGs shown within CpG islands were all typically hypomethylated across all cell and tissue types depicted.
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pone-0111756-g007: High density methylation data derived from bisulfite sequencing of a CATCH-Seq target.Scale of the captured region is indicated in the topmost track in kilobases (kb), followed by repeat structure in gray and black (RepMask), genes shown in blue (RefSeq), and CpG islands in green. Four CATCH-Seq tracks from the same cell type show DNA methylation levels across ∼2,700 target CpGs with hypomethylation depicted in green and hypermethylation in red. Six reduced representation bisulfite sequencing (RRBS) tracks for different cell and tissue types correspond with the same captured region, and demonstrate CpGs not covered by RRBS method compared to CATCH-Seq. The four CATCH-Seq tracks are from the same cell type as the topmost RRBS track. RRBS tracks are derived from previously reported data [23]. CpGs shown within CpG islands were all typically hypomethylated across all cell and tissue types depicted.

Mentions: We have also applied CATCH-Seq to analysis of DNA methylation across large target sites containing CpG islands and multiple genes. By using methylated adapters and treating the post-capture libraries with bisulfite conversion, we can measure CpG methylation with higher coverage thresholds than what is often generated for genome-wide analysis of individual CpGs such as by reduced representation bisulfite sequencing (RRBS) (Figure 7). Furthermore, we have found that CATCH-Seq can also be used in parallel with any other practiced functional genomics approaches. A similar method to ours performed sequencing of BAC-enriched mononucleosomal fragments (known as BEM-Seq), using whole BAC labelled probes for capture of MNAse-digested fragments within a target site [17]. We have similarly used CATCH-Seq procedures with MNase-digests also from sorted mouse lymphocytes in combination with methylation analysis (unpublished). Lastly, CATCH-Seq was also used to capture gene regions associated with melanism from genomes of unsequenced Felid species using selected fosmids from Felis catus as templates [22] (manuscript submitted). Overall, we find that data from CATCH-Seq procedures allows for affordable, high resolution sequencing of captured genomic targets without the added cost of oligo-based probe synthesis. We have included a price per sample estimation with comparison of current commercially available custom probe synthesis platforms for two of our targets we captured (Table S2).


Targeted sequencing of large genomic regions with CATCH-Seq.

Day K, Song J, Absher D - PLoS ONE (2014)

High density methylation data derived from bisulfite sequencing of a CATCH-Seq target.Scale of the captured region is indicated in the topmost track in kilobases (kb), followed by repeat structure in gray and black (RepMask), genes shown in blue (RefSeq), and CpG islands in green. Four CATCH-Seq tracks from the same cell type show DNA methylation levels across ∼2,700 target CpGs with hypomethylation depicted in green and hypermethylation in red. Six reduced representation bisulfite sequencing (RRBS) tracks for different cell and tissue types correspond with the same captured region, and demonstrate CpGs not covered by RRBS method compared to CATCH-Seq. The four CATCH-Seq tracks are from the same cell type as the topmost RRBS track. RRBS tracks are derived from previously reported data [23]. CpGs shown within CpG islands were all typically hypomethylated across all cell and tissue types depicted.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214737&req=5

pone-0111756-g007: High density methylation data derived from bisulfite sequencing of a CATCH-Seq target.Scale of the captured region is indicated in the topmost track in kilobases (kb), followed by repeat structure in gray and black (RepMask), genes shown in blue (RefSeq), and CpG islands in green. Four CATCH-Seq tracks from the same cell type show DNA methylation levels across ∼2,700 target CpGs with hypomethylation depicted in green and hypermethylation in red. Six reduced representation bisulfite sequencing (RRBS) tracks for different cell and tissue types correspond with the same captured region, and demonstrate CpGs not covered by RRBS method compared to CATCH-Seq. The four CATCH-Seq tracks are from the same cell type as the topmost RRBS track. RRBS tracks are derived from previously reported data [23]. CpGs shown within CpG islands were all typically hypomethylated across all cell and tissue types depicted.
Mentions: We have also applied CATCH-Seq to analysis of DNA methylation across large target sites containing CpG islands and multiple genes. By using methylated adapters and treating the post-capture libraries with bisulfite conversion, we can measure CpG methylation with higher coverage thresholds than what is often generated for genome-wide analysis of individual CpGs such as by reduced representation bisulfite sequencing (RRBS) (Figure 7). Furthermore, we have found that CATCH-Seq can also be used in parallel with any other practiced functional genomics approaches. A similar method to ours performed sequencing of BAC-enriched mononucleosomal fragments (known as BEM-Seq), using whole BAC labelled probes for capture of MNAse-digested fragments within a target site [17]. We have similarly used CATCH-Seq procedures with MNase-digests also from sorted mouse lymphocytes in combination with methylation analysis (unpublished). Lastly, CATCH-Seq was also used to capture gene regions associated with melanism from genomes of unsequenced Felid species using selected fosmids from Felis catus as templates [22] (manuscript submitted). Overall, we find that data from CATCH-Seq procedures allows for affordable, high resolution sequencing of captured genomic targets without the added cost of oligo-based probe synthesis. We have included a price per sample estimation with comparison of current commercially available custom probe synthesis platforms for two of our targets we captured (Table S2).

Bottom Line: Furthermore, libraries constructed with methylated adapters prior to solution hybridization also enable targeted bisulfite sequencing.We applied CATCH-Seq to diverse targets ranging in size from 125 kb to 3.5 Mb.Given its similarity in procedure, CATCH-Seq can also be performed in parallel with commercial systems.

View Article: PubMed Central - PubMed

Affiliation: HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, United States of America.

ABSTRACT
Current target enrichment systems for large-scale next-generation sequencing typically require synthetic oligonucleotides used as capture reagents to isolate sequences of interest. The majority of target enrichment reagents are focused on gene coding regions or promoters en masse. Here we introduce development of a customizable targeted capture system using biotinylated RNA probe baits transcribed from sheared bacterial artificial chromosome clone templates that enables capture of large, contiguous blocks of the genome for sequencing applications. This clone adapted template capture hybridization sequencing (CATCH-Seq) procedure can be used to capture both coding and non-coding regions of a gene, and resolve the boundaries of copy number variations within a genomic target site. Furthermore, libraries constructed with methylated adapters prior to solution hybridization also enable targeted bisulfite sequencing. We applied CATCH-Seq to diverse targets ranging in size from 125 kb to 3.5 Mb. Our approach provides a simple and cost effective alternative to other capture platforms because of template-based, enzymatic probe synthesis and the lack of oligonucleotide design costs. Given its similarity in procedure, CATCH-Seq can also be performed in parallel with commercial systems.

Show MeSH