Accurate detection of subclonal single nucleotide variants in whole genome amplified and pooled cancer samples using HaloPlex target enrichment

Abstract

BackgroundTarget enrichment and resequencing is a widely used approach for identification of cancer genes and genetic variants associated with diseases. Although cost effective compared to whole genome sequencing, analysis of many samples constitutes a significant cost, which could be reduced by pooling samples before capture. Another limitation to the number of cancer samples that can be analyzed is often the amount of available tumor DNA. We evaluated the performance of whole genome amplified DNA and the power to detect subclonal somatic single nucleotide variants in non-indexed pools of cancer samples using the HaloPlex technology for target enrichment and next generation sequencing.ResultsWe captured a set of 1528 putative somatic single nucleotide variants and germline SNPs, which were identified by whole genome sequencing, with the HaloPlex technology and sequenced to a depth of 792–1752. We found that the allele fractions of the analyzed variants are well preserved during whole genome amplification and that capture specificity or variant calling is not affected. We detected a large majority of the known single nucleotide variants present uniquely in one sample with allele fractions as low as 0.1 in non-indexed pools of up to ten samples. We also identified and experimentally validated six novel variants in the samples included in the pools.ConclusionOur work demonstrates that whole genome amplified DNA can be used for target enrichment equally well as genomic DNA and that accurate variant detection is possible in non-indexed pools of cancer samples. These findings show that analysis of a large number of samples is feasible at low cost, even when only small amounts of DNA is available, and thereby significantly increases the chances of indentifying recurrent mutations in cancer samples.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-856) contains supplementary material, which is available to authorized users.

Genetics of Pancreatitis: The 2014 Update

“…Targeted capture of custom-designed regions followed by sequencing of selected genomic regions of interest provides an attractive, cost-effective alternative. HaloPlex TM target enrichment technology is a selective circularization-based method that is a further development of the principle of selector probes (Berglund et al 2013). In the HaloPlex TM technology, genomic DNA is fragmented by restriction enzyme digestion and circularized by hybridization to probes whose ends are complementary to the target fragments.…”

MotorPlex provides accurate variant detection across large muscle genes both in single myopathic patients and in pools of DNA samples

“…We started using 200ng of genomic DNA and strictly followed the protocol, with the exception that restricted fragments were hybridized for at least [16][17][18][19][20][21][22][23][24] hours to the specific probes. After the capture of biotinylated target DNA, using streptavidin beads, nicks in the circularized fragments were closed by a ligase.…”

“…Innovative strategies of clinical exome sequencing at high coverage have been described [17], but the cost for a single patient is still too high for routine diagnosis. Thus, there is still space for targeted strategies [18] and the HaloPlex Target Enrichment System [19] represents an innovative technology for targeting, since it uses a combination of eight different enzyme restriction followed by probe capture. It permits a single-tube target amplification and one can accurately predict the precise sequence coverage in advance.…”

Targeted Next-Generation Sequencing Effectively Analyzed the Cystic Fibrosis Transmembrane Conductance Regulator Gene in Pancreatitis

“…This strategy has allowed identification of causal variants in several Mendelian disorders, variants associated with complex diseases, and recurrently mutated cancer genes [18][19][20]. The HaloPlex target enrichment technology is a selective circularization-based method that is a further development of the principle of selector probes [21]. In the HaloPlex technology, genomic DNA is fragmented by restriction enzyme digestion and circularized by hybridization to probes whose ends are complementary to the target fragments.…”