Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer

Abstract

Microsatellites are polymorphic short tandem repeats of 1–6 nucleotides ubiquitously present in the genome that are extensively used in living organisms as genetic markers and in oncology to detect microsatellite instability (MSI). While the standard analysis method of microsatellites is based on PCR followed by capillary electrophoresis, it generates undesirable frameshift products known as ‘stutter peaks’ caused by the polymerase slippage that can greatly complicate the analysis and interpretation of the data. Here we present an easy multiplexable approach replacing PCR that is based on low temperature isothermal amplification using recombinase polymerase amplification (LT-RPA) that drastically reduces and sometimes completely abolishes the formation of stutter artifacts, thus greatly simplifying the calling of the alleles. Using HT17, a mononucleotide DNA repeat that was previously proposed as an optimal marker to detect MSI in tumor DNA, we showed that LT-RPA improves the limit of detection of MSI compared to PCR up to four times, notably for small deletions, and simplifies the identification of the mutant alleles. It was successfully applied to clinical colorectal cancer samples and enabled detection of MSI. This easy-to-handle, rapid and cost-effective approach may deeply improve the analysis of microsatellites in several biological and clinical applications.

Approaches to Sequence the HTT CAG Repeat Expansion and Quantify Repeat Length Variation

“…Some of the limitations of bulk-PCR may be overcome by using recombinase polymerase amplification, an isothermal replacement to PCR that has been shown to produce fewer HTT CAG slippage pr-oducts [ 56 ], or SP-PCR-sequencing which has the potential of allowing the detection of rare large somatic expansions [ 51 ]. However, these approaches are still limited to some extent by PCR slippage, confounding the quantification of somatic contractions ( Table 2 ).…”

“…More accurately quantifying the full range of somatic repeat length changes could also prove useful in identifying genetic modifiers of somatic instability (it is likely that some variants may act to modify the size and direction of repeat length changes, and not just their absolute frequency), and for the development of outcome measures in clinical trials that aim to suppress somatic repeat expansions and/or induce contractions [55]. Some of the limitations of bulk-PCR may be overcome by using recombinase polymerase amplification, an isothermal replacement to PCR that has been shown to produce fewer HTT CAG slippage products [56], or SP-PCR-sequencing which has the potential of allowing the detection of rare large somatic expansions [51]. However, these approaches are still limited to some extent by PCR slippage, confounding the quantification of somatic contractions (Table 2).…”

NGS-based identification and tracing of microsatellite instability from minute amounts DNA using inter-Alu-PCR

“…Similar to other MSI-calling algorithms ( 24 ), for MSI-tracer a minimum sequence coverage of at least 20 for interrogated poly-A target was found necessary to provide reproducible statistics for reliable analysis. Application of experimental approaches to reduce PCR stutter ( 39 , 40 ) could be anticipated to further enhance the reported LODs for MSI detection using MSI-tracer.…”

A New String Edit Distance and Applications

“…To reflect this, a generalization of string edit distance was used to capture similarity between sequences in MPS output. The goal was to include the addition or deletion of a motif as an edit type with a cost that can vary independently from single-nucleotide edits, to reflect the fact that these motif losses and expansions (called backward and forward stutter, respectively) occur consistently across various in vitro applications [18]. The algorithm is also applicable to in vivo methods such as rare disease diagnosis and cancer progression tracking that use STR markers including their stutter [19].…”