Abstract
Solution hybridization capture methods utilize biotinylated oligonucleotides as baits to enrich homologous sequences from next generation sequencing (NGS) libraries. Coupled with NGS, the method generates kilo to gigabases of high confidence consensus targeted sequence. However, in many experiments, a non-negligible fraction of the resulting sequence reads are not homologous to the bait. We demonstrate that during capture, the bait-hybridized library molecules add additional flanking library sequences iteratively, such that baits limited to targeting relatively short regions (e.g. few hundred nucleotides) can result in enrichment across entire mitochondrial and bacterial genomes. Our findings suggest that some of the off-target sequences derived in capture experiments are non-randomly enriched, and that CapFlank will facilitate targeted enrichment of large contiguous sequences with minimal prior target sequence information.
Highlights
Hybridization capture methods are variable in methodology and application though the underlying principles are general [1,2,3,4,5]
Subsequent de novo assembly of the sequence reads that did not map to the control region yielded the remaining mitochondrial genome for each species. (Figure 1 Boxed graphic, Table 1)
Blocking oligonucleotides are added during hybridization to prevent the adaptors from creating unwanted homology during hybridization [9]
Summary
Hybridization capture methods are variable in methodology and application though the underlying principles are general [1,2,3,4,5]. Oligonucleotides are synthesized or PCR products are generated as baits for genomic targets of interest and either affixed to a microarray or biotinylated and bound to streptavidin coated magnetic beads for solution based capture. Regardless of how they are prepared, the bound nucleic acids serve as bait for capturing homologous DNA fragments from a DNA library. Homologous DNA fragments from a generation sequence (NGS) library (e.g. GS FLX or Illumina) that match the bait sequence serve as targets. After purification of the target enriched library, DNA fragments with homology to the baits will be enriched, and nontargeted sequences will have been removed.
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