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

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...

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Autores principales: Shestak, Anna G., Bukaeva, Anna A., Saber, Siamak, Zaklyazminskaya, Elena V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901947/
https://www.ncbi.nlm.nih.gov/pubmed/33633783
http://dx.doi.org/10.3389/fgene.2021.620337
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author Shestak, Anna G.
Bukaeva, Anna A.
Saber, Siamak
Zaklyazminskaya, Elena V.
author_facet Shestak, Anna G.
Bukaeva, Anna A.
Saber, Siamak
Zaklyazminskaya, Elena V.
author_sort Shestak, Anna G.
collection PubMed
description 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.
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spelling pubmed-79019472021-02-24 Allelic Dropout Is a Common Phenomenon That Reduces the Diagnostic Yield of PCR-Based Sequencing of Targeted Gene Panels Shestak, Anna G. Bukaeva, Anna A. Saber, Siamak Zaklyazminskaya, Elena V. Front Genet Genetics 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. Frontiers Media S.A. 2021-02-01 /pmc/articles/PMC7901947/ /pubmed/33633783 http://dx.doi.org/10.3389/fgene.2021.620337 Text en Copyright © 2021 Shestak, Bukaeva, Saber and Zaklyazminskaya. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genetics
Shestak, Anna G.
Bukaeva, Anna A.
Saber, Siamak
Zaklyazminskaya, Elena V.
Allelic Dropout Is a Common Phenomenon That Reduces the Diagnostic Yield of PCR-Based Sequencing of Targeted Gene Panels
title Allelic Dropout Is a Common Phenomenon That Reduces the Diagnostic Yield of PCR-Based Sequencing of Targeted Gene Panels
title_full Allelic Dropout Is a Common Phenomenon That Reduces the Diagnostic Yield of PCR-Based Sequencing of Targeted Gene Panels
title_fullStr Allelic Dropout Is a Common Phenomenon That Reduces the Diagnostic Yield of PCR-Based Sequencing of Targeted Gene Panels
title_full_unstemmed Allelic Dropout Is a Common Phenomenon That Reduces the Diagnostic Yield of PCR-Based Sequencing of Targeted Gene Panels
title_short Allelic Dropout Is a Common Phenomenon That Reduces the Diagnostic Yield of PCR-Based Sequencing of Targeted Gene Panels
title_sort allelic dropout is a common phenomenon that reduces the diagnostic yield of pcr-based sequencing of targeted gene panels
topic Genetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901947/
https://www.ncbi.nlm.nih.gov/pubmed/33633783
http://dx.doi.org/10.3389/fgene.2021.620337
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