Nested Patch PCR enables highly multiplexed mutation discovery in candidate genes

Abstract

Medical resequencing of candidate genes in individual patient samples is becoming increasingly important in the clinic and in clinical research. Medical resequencing requires the amplification and sequencing of many candidate genes in many patient samples. Here we introduce Nested Patch PCR, a novel method for highly multiplexed PCR that is very specific, can sensitively detect SNPs and mutations, and is easy to implement. This is the first method that couples multiplex PCR with sample-specific DNA barcodes and next-generation sequencing to enable highly multiplex mutation discovery in candidate genes for multiple samples in parallel. In our pilot study, we amplified exons from colon cancer and matched normal human genomic DNA. From each sample, we successfully amplified 96% (90 of 94) targeted exons from across the genome, totaling 21.6 kbp of sequence. Ninety percent of all sequencing reads were from targeted exons, demonstrating that Nested Patch PCR is highly specific. We found that the abundance of reads per exon was reproducible across samples. We reliably detected germline SNPs and discovered a colon tumor specific nonsense mutation in APC, a gene causally implicated in colorectal cancer. With Nested Patch PCR, candidate gene mutation discovery across multiple individual patient samples can now utilize the power of second-generation sequencing.

Molecular Evolutionary Analysis of Cancer Cell Lines

“…Furthermore, by screening cell lines from different tissues of origin that might cluster together because of similar DNA variation patterns, new indications for novel drugs can be discovered preclinically. Finally, sophisticated DNA-based diagnostic tests to identify specific target gene mutations are being increasingly used to develop clinical treatment regimens for cancer patients (49). As these data are collected across more genes and patients, evolutionary approaches can be used to classify clinical tumor samples based on molecular similarities and potentially guide therapeutic decisions.…”

Review of massively parallel DNA sequencing technologies

“…This can equally be used for preparation of targets for NGS but owing to the massive capacity of these platforms very large numbers of PCRs are required to fill a run. The processing required can be limited by utilising multiplex PCR or long range PCR Varley and Mitra 2008). Commercial solutions to this problem include the RainStorm technology from RainDance Technologies which uses an emPCR approach to simultaneously amplify up to 4,000 short DNA sequences in separate microdroplets (Tewhey et al 2009), and the Access Array from Fluidigm which uses proprietary microfluidics to setup an array of 2,304 PCRs (48 samples 9 48 assays).…”

Direct multiplex sequencing (DMPS)—a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA

“…Despite these efforts, only small sets of primers could eventually be used in each multiplex amplification reaction (below 10 pairs). Two other studies achieved a higher level of multiplexing by introducing a second round of target selection after amplification, either by selective circularization or by target-specific adapter ligation (Varley and Mitra 2008). However, this requires more reaction steps and the synthesis of additional long oligos for each target.…”

“…Previous studies with modern high-quality DNA have shown that this is in principle possible Varley and Mitra 2008;Goossens et al 2009), but the proposed methods require cost-or time-consuming measures to avoid sequencing PCR artifacts. In one study, all primers were computationally checked for self-dimer and cross-dimer interactions, and the concentration of each primer was optimized in singleplex PCRs (Goossens et al 2009).…”

“…However, this requires more reaction steps and the synthesis of additional long oligos for each target. Furthermore, these methods require modified multiplex PCR primers (Varley and Mitra 2008), produce concatemeric products ), or are not easily compatible with sample barcoding Goossens et al 2009). …”