Allelic Dropout Is a Common Phenomenon That Reduces the Diagnostic Yield of PCR-Based Sequencing of Targeted Gene Panels

Abstract

Primary cardiomyopathies (CMPs) are monogenic but multi-allelic disorders with dozens of genes involved in pathogenesis. The implementation of next-generation sequencing (NGS) approaches has resulted in more time- and cost-efficient DNA diagnostics of cardiomyopathies. However, the diagnostic yield of genetic testing for each subtype of CMP fails to exceed 60%. The aim of this study was to demonstrate that allelic dropout (ADO) is a common phenomenon that reduces the diagnostic yield in primary cardiomyopathy genetic testing based on targeted gene panels assayed on the Ion Torrent platform. We performed mutational screening with three custom targeted gene panels based on sets of oligoprimers designed automatically using AmpliSeq Designer® containing 1049 primer pairs for 37 genes with a total length of 153 kb. DNA samples from 232 patients were screened with at least one of these targeted gene panels. We detected six ADO events in both IonTorrent PGM (three cases) and capillary Sanger sequencing (three cases) data, identifying ADO-causing variants in all cases. All ADO events occurred due to common or rare single nucleotide variants (SNVs) in the oligoprimer binding sites and were detected because of the presence of “marker” SNVs in the target DNA fragment. We ultimately identified that PCR-based NGS involves a risk of ADO that necessitates the use of Sanger sequencing to validate NGS results. We assume that oligoprimer design without ADO data affects the amplification efficiency of up to 0.77% of amplicons.

Targeted Sequencing of 242 Clinically Important Genes in the Russian Population From the Ivanovo Region

“…Moreover, this whole-exome study demonstrated the overrepresentation of several disease-causing variants for Mendelian disorders, such as phenylketonuria (PAH, rs5030858), Wilson's disease (ATP7B, rs76151636), factor VII deficiency (F7, rs36209567), and the kyphoscoliosis type of Ehlers-Danlos syndrome (FKBP14, rs542489955). For the Russian population, however, pathogenic variant frequencies were reported mostly for relatively small cohorts including patients and their families and targeted at specific genes and disorders, for example, familial hypercholesterolemia (Meshkov et al, 2021;Miroshnikova et al, 2021); cystic fibrosis, phenylketonuria, alpha-1 antitrypsin deficiency, and sensorineural hearing loss (Kiseleva et al, 2020;Petrova et al, 2020); cardiomyopathy (Marakhonov et al, 2019;Zaklyazminskaya et al, 2019;Kulikova et al, 2021;Shestak et al, 2021); and breast and ovarian cancer (Brovkina et al, 2018;Solodskikh et al, 2019).…”

Coexistence of Two Rare Genetic Variants in Canonical and Non-canonical Exons of SCN5A: A Potential Source of Misinterpretation

“…Machine-learning methods can also improve significantly recognition of the regulatory sequence patterns, splice sites placed in deep intronic areas, functional annotation of the genes, and co-functional relationship of the gene products. Complementary to the data interpretation, AI-based algorithms might better elicit technical artifacts and other phenomena, such as allelic drop-out, that reduce diagnostic yield of genetic testing ( Shestak et al, 2021 ).…”

Tumor BRCA testing in ovarian cancer and EQA scheme: our experience of a critical evaluation

“…ADO is a common phenomenon that reduces the efficiency of PCR-based targeted sequencing. It was first described as a "partial amplification failure" causing a potential source of misdiagnosis for both dominant and recessive diseases [12]. The ADO phenomenon involves selective allele amplification during the PCR process.…”