Increasing the sensitivity of low-level mutations using COLD-PCR

The lower limit of sensitivity using next-generation sequencing is generally accepted to be 5%. The reason for this detection limit has to do with the error rate of the platform, rather than as a function of base coverage read depth.

A graphical illustration of this sensitivity limit using the SOLiD^®Sequencing System is still available online, as an interactive flash-based graphic. The Variant Frequency in Hetergeneous Sample is a horizontal slider, and top ‘Coverage Required for Detection of Variants at 05% Frequency in a Hetergenous Sample’ reads 99% accuracy, 450x coverage. At 98% accuracy, a 5% minor allele goes undetected. (Here’s the link to the calculator.)

Minor variants are single nucleotide polymorphisms (SNPs) that present as a minor component with a contribution of less than 25% at a given allele. For minor allele detection via Sanger Sequencing, the generally accepted lower limit of detection is on the order of 20%. But with the new Applied Biosystems™ Minor Variant Finder Software developed for the detection and reporting of minor mutations by Sanger sequencing,able to detect 5% minor variants at a sensitivity above 96% and specificity above 99.8%.

A method called CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) was first introduced in 2008 and increases the sensitivity of Sanger Sequencing or other genotyping (including NGS) assays. COLD-PCR enriches the minor alleles preferentially during PCR, allowing low-frequency mutations to be represented at a much higher threshold for detection (i.e. in the context of NGS greater than 5%).

It works on the physical basis of melting temperature (Tm) of a given stretch of a targeted amplicon. A single base change can make the Tm shift in the range of 0.2-1.5C, up to 200bp long (for details, here’s a 2004 reference). The technique uses a ‘critical denaturation temperature’ Tc, which is below Tm at which PCR efficiency drops abruptly.

Thus using an intermediate step in PCR cycling conditions, where mutant DNA will remain denatured but at which wild-type DNA is double-stranded, is the mutant DNA available for preferential amplification.

Recently this approach has been further extended to the Ion AmpliSeq™ Panel amplicon-based enrichment method, as well as for conventional Sanger Sequencing.The authors not only use a change in denaturation temperature, but also use alternative nucleotide analogs (dITP and dDTP) to enable detection of not only Tm-lowering mutations but also Tm-increasing ones to enrich for the maximum mutation types.

The paper, entitled ‘Single-tube, highly parallel mutation enrichment in cancer gene panels by use of temperature-tolerant COLD-PCR’using research panels showed results using both the Ion AmpliSeq™ Cancer Hotspot v2 and the Ion AmpliSeq™ Comprehensive Cancer Panel down to 0.01-0.1% sensitivity, and single amplicon sequencing of TP53 individual exons via Sanger Sequencing down to 0.1% – 0.3%.