Abstract
BackgroundTargeted sequencing is a powerful tool with broad application in both basic and translational sciences. Relatively low on-target rates for most current targeted sequencing studies influence the required coverage and data quality for subsequent applications.ResultsWe present an improved targeted sequencing method that uses two rounds of in solution hybridization with probes synthesized from genomic clone templates, termed dCATCH-Seq. Independent captures of two large continuous genomic regions across three cell types within the human major histocompatibility complex (MHC) that spans ~3.5 Mb and a ~250 kb region on chromosome 11 demonstrated that dCATCH-Seq was highly reproducible with ~95% capture specificity. Comprehensive analyses of sequencing data generated using the dCATCH-Seq approach also showed high accuracy in the detection of genetic variants and HLA typing. The double hybridization capture approach can also be coupled with bisulfite sequencing for DNA methylation profiling of both CpG and non-CpG sites.ConclusionsAltogether, dCATCH-Seq is a powerful and scalable targeted sequencing approach to investigate both genetic and epigenetic features.
Highlights
Targeted sequencing is a powerful tool with broad application in both basic and translational sciences
Following a protocol presented in the previous CATCH-seq, but undergoing two rounds of hybridization capture, we referred to this strategy as dCATCH-Seq
A total of 170,700 and 140,200 reads on average were obtained across technical replicates for the single and double hybridizations, respectively, and on average 96.3% of the reads were mapped to the reference genome (Additional file 1: Table S1)
Summary
Targeted sequencing is a powerful tool with broad application in both basic and translational sciences. Low on-target rates for most current targeted sequencing studies influence the required coverage and data quality for subsequent applications. The primary design of targeted sequencing is to capture genetic variants within intended regions [1] Some targets, such as exome capture, has been broadly used in both basic and translational research that includes characterization of genetic diversity and demographic history in human populations [2, 3], identification of etiological variants [4], crossspecies genome comparisons [5], and even phylogenetic estimation [6]. There are three main techniques [7] for targeted enrichment of DNA sequences that include hybridizationbased capture either in solution or on a solid support, e.g., TruSeq (Illumina), SureSelect (Agilent), and SeqCap (Roche NimbleGen) platforms [8,9,10], selective circularization, and PCR amplification. Some strategies in measuring target efficiency have been developed, e.g., SeqCap qPCR kit (Roche) and Multipoint Test for Targeted-enrichment Efficiency (MTTE) [17]
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