Single-Tube, Highly Parallel Mutation Enrichment in Cancer Gene Panels by Use of Temperature-Tolerant COLD-PCR

Abstract

BACKGROUND Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-re-sequencing. Analysis of mutations below ~2–5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids, is problematic due to increased ‘noise’ from sequencing errors. Technologies that reduce noise via deep-sequencing unavoidably reduce throughput and increase cost. Here we provide proof-of-principle that COLD-PCR technology enables multiplex low-level mutation detection in cancer gene panels while retaining throughput. METHODS We have developed a multiplex temperature-tolerant-COLD-PCR (fast-TT-COLD-PCR) approach that uses cancer gene panels developed for massively parallel sequencing. Following a multiplex pre-amplification from genomic DNA we attach tails to all amplicons and perform fast-TT-COLD-PCR. This approach gradually increases denaturation temperatures in a step-wise fashion, such that all possible denaturation temperatures are encompassed. By introducing modified nucleotides, fast-COLD-PCR is adapted to enrich for Tm-increasing as well as Tm-decreasing mutations over all amplicons, in a single tube. RESULTS Using custom-made and commercial gene panels containing 8, 50, 190 or 16,000 amplicons we demonstrate that fast-TT-COLD-PCR enriches mutations on all examined targets simultaneously. Incorporation of dITP/dDTP in place of dGTP/dATP enables enrichment of Tm-increasing mutations. Serial dilution experiments demonstrate a limit-of-detection of ~ 0.01–0.1% mutation abundance using Ion-Torrent and 0.1–0.3% using Sanger sequencing. CONCLUSIONS Fast-TT-COLD-PCR improves the limit of detection of cancer gene panels by enabling mutation enrichment in multiplex, single tube reactions. This novel adaptation of COLD-PCR converts subclonal mutations to clonal, thereby facilitating detection and subsequent mutation sequencing.

Sensitive Detection of DNA Lesions by Bulge-Enhanced Highly Specific Coamplification at Lower Denaturation Temperature Polymerase Chain Reaction

“…For both types of the lesions, the C t value shows good linear relation with the logarithm of the fraction of lesion-containing DNA in a broad range of 0.01–100% (Figure , parts B and D). To the best of our knowledge, this is the lowest abundance of single-nucleotide change that can be reached by COLD-PCR without any auxiliary probe and downstream analysis. , …”

“…To the best of our knowledge, this is the lowest abundance of single-nucleotide change that can be reached by COLD-PCR without any auxiliary probe and downstream analysis. 32,33 Glycosylase plays a crucial role in the BER pathway. 34 The expression level of glycosylase is associated with various diseases including inflammation, 35 Alzheimer's disease, 36 and cancers.…”

Multiplexing of E-ice-COLD-PCR Assays for Mutation Detection and Identification

“…Therefore, different full-, fast-, or ice-COLD-PCR assays might be less suitable for working under a single thermocycling program and even less suitable to multiplexing. For the specific case of Tm-reducing mutations (where Tm is the temperature at which 50% of double-stranded DNA or DNA/RNA hybrids is denatured) (C:GϾA:T or ϾT:A), which represent 60%-70% of all mutations in cancer, these issues have been addressed by the development of temperature-tolerant (TT)-COLD-PCR, which relies on the use of a Tc incremented by 0.3-0.5°C for each defined number of cycles, leading to mutation enrichment when the Tc falls in the right temperature window (5,6 ).…”

Highly Sensitive Enrichment of Low-Frequency Variants by Hairpin Competition Amplification

“…12,13 However, due to abundant wild template backgrounds, rare single-nucleotide variants are difficult to detect effectively with high sensitivity, which presents a challenge to clinical and diagnostic applications. 14,15 A large number of allele-specific polymerase chain reaction (PCR) approaches for identifying known rare variants have been developed, including amplification refractory mutation system (ARMS), 16 allele-specific PCR with a blocking reagent (ASB-PCR), 17 PCR by toehold hairpin primers, 18 PNAmediated PCR clamping, 19,20 use of a snapback primer based on a melting curve, 21 co-amplification-at-lower-denaturationtemperature PCR (COLD-PCR), 22,23 single-nucleotide-specific programmable riboregulators (SNIPRs), 24 and digital PCR (dPCR) platform. 25,26 However, most of these genotyping methods require specific modified bases, use complex performing protocols, have limited sensitivity, require special instrument sets, or are restricted by multiplex capacity.…”