Scientists at the University of Oregon have published a new method to detect PCR and sequencing errors that should help other researchers track rare SNPs with greater accuracy. PELE-seq, which gets our vote for best new protocol name, can be used with ddRAD-seq, targeted amplicon sequencing, and many other genotyping methods.
From lead author Jessica Preston and senior author Eric Johnson, “High-specificity detection of rare alleles with Paired-End Low Error Sequencing (PELE-Seq)” came out in BMC Genomics. The scientists embarked on this project to reduce the current error rate in NGS studies, which they peg at about 1% and say “leads to the generation of millions of sequencing errors in a single experiment.”
The team uses barcoded adapters as well as overlapping paired-end reads on size-selected DNA molecules to maximize accuracy. The barcoding process reduces false-positive SNP calls, while the overlapping reads reduce sequencing errors. The team used our Pippin Prep automated DNA sizing platform to collect tight DNA bands prior to paired-end
sequencing on Illumina. Scientists tested the PELE-seq protocol on E. coli and Caenorhabditis remanei, finding improved specificity and sensitivity for accurately detecting rare variants.
“We have demonstrated that the PELE-Seq method of variant calling is highly specific at detecting rare SNPs found at below 1% in a population,” the scientists write. “There were zero instances of false positive SNPs called from PELE-sequenced control E. coli libraries containing rare alleles present at known frequencies, whereas standard NGS DNA-Seq libraries contained 30–50% false-positive SNPs.”