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

Abstract

BackgroundThe 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 of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kbp. The DNA enrichment methodology described herein, Region-Specific Extraction (RSE), produces DNA segments in excess of 20 kbp in length. Coupling this enrichment method to appropriate sequencing platforms will significantly enhance the ability to generate complete and accurate sequence characterization of any genomic region without the need for reference-based assembly.ResultsRSE is a long-range DNA target capture methodology that relies on the specific hybridization of short (20-25 base) oligonucleotide primers to selected sequence motifs within the DNA target region. These capture primers are then enzymatically extended on the 3’-end, incorporating biotinylated nucleotides into the DNA. Streptavidin-coated beads are subsequently used to pull-down the original, long DNA template molecules via the newly synthesized, biotinylated DNA that is bound to them. We demonstrate the accuracy, simplicity and utility of the RSE method by capturing and sequencing a 4 Mbp stretch of the major histocompatibility complex (MHC). Our results show an average depth of coverage of 164X for the entire MHC. This depth of coverage contributes significantly to a 99.94 % total coverage of the targeted region and to an accuracy that is over 99.99 %.ConclusionsRSE represents a cost-effective target enrichment method capable of producing sequencing templates in excess of 20 kbp in length. The utility of our method has been proven to generate superior coverage across the MHC as compared to other commercially available methodologies, with the added advantage of producing longer sequencing templates amenable to DNA sequencing on recently developed platforms. Although our demonstration of the method does not utilize these DNA sequencing platforms directly, our results indicate that the capture of long DNA fragments produce superior coverage of the targeted region.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2836-6) contains supplementary material, which is available to authorized users.

The soybean GmSNAP18 gene underlies two types of resistance to soybean cyst nematode

“…RSE has been successfully applied to yeast, human and zebrafish1112131415, but has not yet been applied to plants. Here we show that RSE-Seq of a targeted 300 kb genomic DNA segment (Gm18: 1480001..1780000) of contrasting chromosomal regions underlying resistance was effective in the direct identification of a candidate rhg1 SCN resistance gene (Table 1 and Fig.…”

“…We then applied ‘region-specific extraction sequencing' (RSE-Seq), a capture technology developed to enrich a targeted chromosomal segment for genome sequencing111213, to identify SCN resistance genes within the identified 300 kb chromosomal segment carrying the rhg1 locus. RSE-Seq has been applied successfully to study yeast, human and zebrafish1112131415. The SNPs and insertions and deletions (InDels) of four soybean lines (Essex, Forrest, Peking and PI 88788) were analysed using the Williams 82 genomic sequence as a reference16.…”

Pharmacogenomic Biomarkers for Improved Drug Therapy—Recent Progress and Future Developments

“…Recent progress towards higher throughput, including whole genome sequencing (WGS), as well as detection of DNA methylation, also makes it well suited for biomarker discovery in complex regions of the genome [125,126]. Furthermore, long-read sequencing combined with target capture methods based on the hybridization of biotinylated baits offers the possibility to focus on specific genomic regions of interest [127,128]. A recent elegant approach demonstrated the utility of direct selection of DNA fragments in real-time by dynamic time warping and matching reads to the reference Components of the epigenetic machinery, such as DNA methyltransferases (DNMTs), histone modifying enzymes (DOT1L, MLLs, KDMs), and nucleosome remodelers are inactivated in up to 50% of ovarian or hepatocellular carcinomas [97,98].…”

CRISPR-assisted targeted enrichment-sequencing (CATE-seq)

“…The first is DNA hybridization on solid surface such as DNA chip or microarray (10)(11)(12)(13)(14)(15). The second is DNA hybridization in solution with biotin-labeled capture probes (DNA or RNA) and the hybrids were isolated with streptavidin-coupled magnetic beads (4,5,16,17). The third is the targeted PCR amplification (8,18,19).…”