Quick, sensitive and specific detection and evaluation of quantification of minor variants by high-throughput sequencing.

摘要: Minor variants have significant implications in quasispecies evolution, early cancer detection and non-invasive fetal genotyping but their accurate by next-generation sequencing (NGS) is hampered errors. We generated data from mixtures at predetermined ratios order to provide insight into errors variations that can arise for which simulation cannot be performed. The information also enables better parameterization depth of coverage, read quality heterogeneity, library preparation techniques, technical repeatability mathematical modeling, theory development experimental design. devised minor variant authentication rules achieved 100% accuracy both testing validation experiments. are free tedious inspection alignment accuracy, or introduced homopolymers. processes only require to: (1) minimum coverage larger than 30; (2) reported (a) four more callers, (b) DiBayes LoFreq, plus SNVer (or BWA when no results returned SNVer), with the interassay coefficient variation (CV) 0.1. Quantification undermined could neither overcome ultra-deep sequencing, nor recruiting callers reach a consensus, such consistent underestimation overestimation (i.e. low CV) were observed. To accommodate stochastic error adjust observed ratio within specified we presented proof concept use double calibration curve quantification, provides an important reference towards potential industrial-scale fabrication calibrants NGS.