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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

The effect of repeat blocking with increased concentrations of Cot-1 DNA within the CATCH-Seq hybridization step of a chromosome 11 target.Total numbers of on target and off target read yields in millions within non-repetitive sequences (A) or repetitive sequences (B). (C–H) On and off target read yields within repeat structures based on different thresholds of size (C,E,G) or divergence (D,F,H). Green and gray lines show on target and off target reads, respectively.
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pone-0111756-g005: The effect of repeat blocking with increased concentrations of Cot-1 DNA within the CATCH-Seq hybridization step of a chromosome 11 target.Total numbers of on target and off target read yields in millions within non-repetitive sequences (A) or repetitive sequences (B). (C–H) On and off target read yields within repeat structures based on different thresholds of size (C,E,G) or divergence (D,F,H). Green and gray lines show on target and off target reads, respectively.

Mentions: To further investigate the effect of repeat blocking on target coverage, we performed solution hybridization reactions with libraries prepared from K562 cell line genomic DNA and increasing Cot-1 DNA concentrations to test the influence of this blocking reagent on repeat coverage in another chromosome 11 target that was also captured for bisulfite sequencing of an independent sample set (Table S1, Figure 4). We typically used a 20∶1 concentration of Cot-1 to library ratio and were interested in how reduction of Cot-1 DNA influenced on-target capture in both repeats and non-repeats. Approximately 50 million reads were sampled with a MAPQ of greater than or equal to 20 from the total yield of aligned reads from each hybridization with 2.5, 5, 10, and 20 fold Cot-1 to library ratio. We found that with lower input concentration of Cot-1 DNA, we compromised overall target capture efficiency in both non-repetitive sites and in repeats. Increasing concentrations of Cot-1 DNA improved both the absolute yield of reads on-target while also decreasing off-target yields (Figure 5). CATCH-Seq procedures with no Cot-1 DNA in the hybridization step yielded less than 9% of mapped reads within a target sites. In reads aligned to non-repeat sequence, we found a stronger relationship between Cot-1 concentrations and increasing on-target reads than with reduced off-target reads (Figure 5A). By comparison, increasing Cot-1 concentration produced a roughly equal exchange of reads aligned to off-target repeats as for those aligned to on-target repeats (Figure 5B). We found that this rate of exchange between off-target and on-target repeats varied depending on repeat size and SW score. Smaller repeats of less than 250 bp exhibited an equal exchange in off-target for on-target reads, while larger repeats and those with higher SW scores showed a mild increase in yield of on-target reads, while off-target yields declined (Figure 5C–H). Overall, the highest Cot-1 concentration at 20 fold produced the highest on-target read yield.


Targeted sequencing of large genomic regions with CATCH-Seq.

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

The effect of repeat blocking with increased concentrations of Cot-1 DNA within the CATCH-Seq hybridization step of a chromosome 11 target.Total numbers of on target and off target read yields in millions within non-repetitive sequences (A) or repetitive sequences (B). (C–H) On and off target read yields within repeat structures based on different thresholds of size (C,E,G) or divergence (D,F,H). Green and gray lines show on target and off target reads, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214737&req=5

pone-0111756-g005: The effect of repeat blocking with increased concentrations of Cot-1 DNA within the CATCH-Seq hybridization step of a chromosome 11 target.Total numbers of on target and off target read yields in millions within non-repetitive sequences (A) or repetitive sequences (B). (C–H) On and off target read yields within repeat structures based on different thresholds of size (C,E,G) or divergence (D,F,H). Green and gray lines show on target and off target reads, respectively.
Mentions: To further investigate the effect of repeat blocking on target coverage, we performed solution hybridization reactions with libraries prepared from K562 cell line genomic DNA and increasing Cot-1 DNA concentrations to test the influence of this blocking reagent on repeat coverage in another chromosome 11 target that was also captured for bisulfite sequencing of an independent sample set (Table S1, Figure 4). We typically used a 20∶1 concentration of Cot-1 to library ratio and were interested in how reduction of Cot-1 DNA influenced on-target capture in both repeats and non-repeats. Approximately 50 million reads were sampled with a MAPQ of greater than or equal to 20 from the total yield of aligned reads from each hybridization with 2.5, 5, 10, and 20 fold Cot-1 to library ratio. We found that with lower input concentration of Cot-1 DNA, we compromised overall target capture efficiency in both non-repetitive sites and in repeats. Increasing concentrations of Cot-1 DNA improved both the absolute yield of reads on-target while also decreasing off-target yields (Figure 5). CATCH-Seq procedures with no Cot-1 DNA in the hybridization step yielded less than 9% of mapped reads within a target sites. In reads aligned to non-repeat sequence, we found a stronger relationship between Cot-1 concentrations and increasing on-target reads than with reduced off-target reads (Figure 5A). By comparison, increasing Cot-1 concentration produced a roughly equal exchange of reads aligned to off-target repeats as for those aligned to on-target repeats (Figure 5B). We found that this rate of exchange between off-target and on-target repeats varied depending on repeat size and SW score. Smaller repeats of less than 250 bp exhibited an equal exchange in off-target for on-target reads, while larger repeats and those with higher SW scores showed a mild increase in yield of on-target reads, while off-target yields declined (Figure 5C–H). Overall, the highest Cot-1 concentration at 20 fold produced the highest on-target read yield.

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