Representation of the allelic dropout phenomenon in the sequencing results

Abstract

All PCR-based sequencing methods have a risk of allelic dropout (ADO) phenomenon, which leads to selective allele amplification during PCR process and may reduce the diagnostic yield of genetic testing. To identify the cases of ADO we compared BAM and VCF files with Sanger sequencing chromatograms. To reveal the causes of ADO, primer binding sites using the gnomAD database were analysed. All amplicons with suspected ADO cases were re-sequenced using the alternative oligoprimers pairs. We have identified 8 cases of ADO both in NGS sequences of targeted genes panels and direct Sanger sequencing. The fact of selective allele amplification was confirmed in all cases by re-sequencing using an alternative pair of primers. Most cases of ADO (6 cases, 75%) were caused by common or rare single nucleotide genetic variants at the annealing sites of oligoprimers, and in two cases (25%) ADO was presumably mediated by the presence of six- or more nucleotides indels in the amplicons studied. In addition, in some amplicons we found the “underrepresentation” of SNVs in NGS reads or the presence of the SNV only in the reads of one amplicon out of two. Given cases of proven and potentially ADO, we suppose that design of oligoprimers without registration of ADO phenomenon may affect the amplification efficiency up to 0,85% of amplicons.