Multiplex Target Enrichment Using DNA Indexing for Ultra-High Throughput SNP Detection

Abstract

Screening large numbers of target regions in multiple DNA samples for sequence variation is an important application of next-generation sequencing but an efficient method to enrich the samples in parallel has yet to be reported. We describe an advanced method that combines DNA samples using indexes or barcodes prior to target enrichment to facilitate this type of experiment. Sequencing libraries for multiple individual DNA samples, each incorporating a unique 6-bp index, are combined in equal quantities, enriched using a single in-solution target enrichment assay and sequenced in a single reaction. Sequence reads are parsed based on the index, allowing sequence analysis of individual samples. We show that the use of indexed samples does not impact on the efficiency of the enrichment reaction. For three- and nine-indexed HapMap DNA samples, the method was found to be highly accurate for SNP identification. Even with sequence coverage as low as 8x, 99% of sequence SNP calls were concordant with known genotypes. Within a single experiment, this method can sequence the exonic regions of hundreds of genes in tens of samples for sequence and structural variation using as little as 1 μg of input DNA per sample.

Efficient Genome-Wide Detection and Cataloging of EMS-Induced Mutations Using Exome Capture and Next-Generation Sequencing

“…Furthermore, we observed that multiplexing enhanced uniformity of capture and sequencing (Supplemental Figure 2) but was not associated with any detectable loss of complexity, at least at the coverage levels that we produced (Supplemental Figures 1 and 4). Multiplexed exome capture has been described before, although either with a smaller number of multiplexed samples (Cummings et al, 2010;Kenny et al, 2011;Wesolowska et al, 2011) or a much smaller targeted space (for example, Rohland and Reich [2012] demonstrated multiplexing with 96 individuals on a 2.2-Mb targeted space). The effectiveness of exome capture for resequencing of ;20 Mb of exon space was also recently demonstrated in Populus trichocarpa, for which multiplexing was used at the level of sequencing but not at the level of the capture reaction (Zhou and Holliday, 2012).…”

Ultraconserved elements are novel phylogenomic markers that resolve placental mammal phylogeny when combined with species-tree analysis

“…Similar probe sets could be designed for other phylogenetic groups (Siepel et al 2005). Increasingly sophisticated hierarchical methods for barcoding individuals (Kenny et al 2011) will enable targeted capture and sequencing of orthologous DNA from many individuals at hundreds or thousands of loci in a partial, massively parallel sequencing run without the laborious intermediate steps of marker discovery, variability screening, individual PCRs, and haplotype phasing. We envision that this approach, when applied to nonmodel organisms, could stimulate a shift in the way researchers collect and analyze broad-scale phylogenetic data, which will build from and complement whole-genome data produced by the Genome 10K Project (http://genome10k.soe.ucsc.edu/).…”

Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes

“…Illumina adapters were replaced by indexed adapters (Eurogentec, Angers, France), previously published by Huentelman's team. 20 The SureSelect enrichment process was performed either before combining indexed samples (hereafter called 'pooling after capture') according to the manufacturer's procedures (Agilent) or after combining equimolarly indexed samples (hereafter called 'pooling before capture') according to Kenny et al 21 The current protocol, available on request, was robotized on two Biomek FX workstations dedicated to the pre-and post-PCR zone. Libraries were then sequenced on GAIIx (Illumina, San Diego, CA, USA) using the paired-end 2 Â 76 bp program.…”