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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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Related in: MedlinePlus

Overview of the clone adapted template capture hybridization sequencing procedure.BAC clone templates are selected to span genomic coordinates of interest, and pooled by percent mass of the composite target. BACs are sheared, ligated with T7 adapters to transcribe biotinylated RNA probes, and then solution hybridized with prepared libraries. Following capture, libraries are amplified by PCR, or bisulfite converted prior to amplification for analysis of DNA methylation. Target enriched libraries are pooled and sequenced.
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pone-0111756-g001: Overview of the clone adapted template capture hybridization sequencing procedure.BAC clone templates are selected to span genomic coordinates of interest, and pooled by percent mass of the composite target. BACs are sheared, ligated with T7 adapters to transcribe biotinylated RNA probes, and then solution hybridized with prepared libraries. Following capture, libraries are amplified by PCR, or bisulfite converted prior to amplification for analysis of DNA methylation. Target enriched libraries are pooled and sequenced.

Mentions: We developed a simple approach for solution hybridization capture sequencing of large genomic targets without the need for oligonucleotide synthesis of target templates (Figure 1). BAC clones were selected across genomic coordinates of interest to generate templates for probe synthesis. PCR or restriction digest was used to first correctly identify selected clones, and BAC DNA was purified. For composite targets of interest greater than what was covered by a single clone, multiple BAC DNAs (contiguous or discontiguous regions) were pooled based on individual percent of composite size in basepairs of the target multiplied by the mass of input template DNA (Figure 1). Pooled BAC template DNA was randomly sheared, ligated with T7 promoter-containing adapters, and T7 forward adapter oligo was annealed to generate double stranded promoter regions with single stranded antisense templates. Following cleanup of the annealing reaction, in vitro transcription was used in the presence of biotin-UTP to synthesize the probes. A similar approach for probe synthesis was also recently described to enrich for ancient human DNA from environmental contaminants using the entire human genome as a template using T7-promoter containing adapters [18]. Solution hybridization and capture procedures were described previously for Illumina libraries, and our protocol is similar except with increased Cot-1 concentration, larger reaction volume, and additional wash steps [5]. We also typically hybridized library samples in 12-plex or 24-plex using inline barcoded adapters, and adjusted hybridization reaction volumes for scaling up concentration of pooled library and hybridization reagents appropriately. Before PCR enrichment of captured library, bisulfite conversion was also used to analyze DNA methylation in regions of interest by use of conversion-resistant, inline barcoded adapters used in library construction.


Targeted sequencing of large genomic regions with CATCH-Seq.

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

Overview of the clone adapted template capture hybridization sequencing procedure.BAC clone templates are selected to span genomic coordinates of interest, and pooled by percent mass of the composite target. BACs are sheared, ligated with T7 adapters to transcribe biotinylated RNA probes, and then solution hybridized with prepared libraries. Following capture, libraries are amplified by PCR, or bisulfite converted prior to amplification for analysis of DNA methylation. Target enriched libraries are pooled and sequenced.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111756-g001: Overview of the clone adapted template capture hybridization sequencing procedure.BAC clone templates are selected to span genomic coordinates of interest, and pooled by percent mass of the composite target. BACs are sheared, ligated with T7 adapters to transcribe biotinylated RNA probes, and then solution hybridized with prepared libraries. Following capture, libraries are amplified by PCR, or bisulfite converted prior to amplification for analysis of DNA methylation. Target enriched libraries are pooled and sequenced.
Mentions: We developed a simple approach for solution hybridization capture sequencing of large genomic targets without the need for oligonucleotide synthesis of target templates (Figure 1). BAC clones were selected across genomic coordinates of interest to generate templates for probe synthesis. PCR or restriction digest was used to first correctly identify selected clones, and BAC DNA was purified. For composite targets of interest greater than what was covered by a single clone, multiple BAC DNAs (contiguous or discontiguous regions) were pooled based on individual percent of composite size in basepairs of the target multiplied by the mass of input template DNA (Figure 1). Pooled BAC template DNA was randomly sheared, ligated with T7 promoter-containing adapters, and T7 forward adapter oligo was annealed to generate double stranded promoter regions with single stranded antisense templates. Following cleanup of the annealing reaction, in vitro transcription was used in the presence of biotin-UTP to synthesize the probes. A similar approach for probe synthesis was also recently described to enrich for ancient human DNA from environmental contaminants using the entire human genome as a template using T7-promoter containing adapters [18]. Solution hybridization and capture procedures were described previously for Illumina libraries, and our protocol is similar except with increased Cot-1 concentration, larger reaction volume, and additional wash steps [5]. We also typically hybridized library samples in 12-plex or 24-plex using inline barcoded adapters, and adjusted hybridization reaction volumes for scaling up concentration of pooled library and hybridization reagents appropriately. Before PCR enrichment of captured library, bisulfite conversion was also used to analyze DNA methylation in regions of interest by use of conversion-resistant, inline barcoded adapters used in library construction.

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
Related in: MedlinePlus