The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity

摘要: The ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. While emerging sequencing platforms are capable producing several kilobases-long reads, fragment sizes generated by current target enrichment technologies remain limiting factor, generally shorter than 1 kbp. methodology described herein, Region-Specific Extraction (RSE), produces segments in excess 20 kbp length. Coupling this method appropriate will significantly enhance generate complete accurate any region without need for reference-based assembly. RSE is long-range that relies on specific hybridization short (20-25 base) oligonucleotide primers selected motifs within region. These then enzymatically extended 3’-end, incorporating biotinylated nucleotides into DNA. Streptavidin-coated beads subsequently used pull-down original, long template molecules via newly synthesized, bound them. We demonstrate accuracy, simplicity utility capturing 4 Mbp stretch major histocompatibility (MHC). Our results show an average depth coverage 164X entire MHC. This contributes 99.94 % total targeted accuracy over 99.99 %. cost-effective templates our has been proven superior across MHC as compared other commercially available methodologies, with added advantage longer amenable recently developed platforms. Although demonstration does not utilize these directly, indicate produce