Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system

BMC Genomics
Gergely IvádyIstván Balogh

Abstract

Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. Although pyro- and ion semiconductor sequencing both have the advantage of delivering long and high quality reads, problems might occur when sequencing homopolymer-containing regions, since the repeating identical bases are going to incorporate during the same synthesis cycle, which leads to uncertainty in base calling. The aim of this study was to evaluate the analytical performance of a pyrosequencing-based next-generation sequencing system in detecting homopolymer sequences using homopolymer-preintegrated plasmid constructs and human DNA samples originating from patients with cystic fibrosis. In the plasmid system average correct genotyping was 95.8% in 4-mers, 87.4% in 5-mers and 72.1% in 6-mers. Despite the experienced low genotyping accuracy in 5- and 6-mers, it was possible to generate amplicons with more than a 90% adequate detection rate in every homopolymer tract. When homopolymers in the CFTR gene were sequenced average accuracy was 89.3%, but varied in a wide range (52.2 - 99.1%). In all but one case, an optimal amplicon-sequencing primer combination could be identified...Continue Reading

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Citations

Dec 8, 2020·Frontiers in Immunology·Mireille Ahou AmiahWilliam Yavo
Sep 23, 2021·Molecular Ecology Resources·Yuanyuan ChengKatherine Belov
Dec 7, 2021·Microbial Genomics·Pakorn AiewsakunArunee Thitithanyanont

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Methods Mentioned

BETA
genotyping
PCR

Software Mentioned

FlowClus
Ensembl
Amplicon Variant Analyzer
Denoiser
DUDE
AmpliconNoise
HECTOR
Seq
Ion Torrent
PyroNoise

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