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Virologica Sinica, 33 (6) : 557, 2018
An Assessment of Amplicon-Sequencing Based Method for Viral Intrahost Analysis
1 Beijing Institute of Radiation Medicine, Beijing 100850, China
2 Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China
3 Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
 Correspondence: liud@wh.iov.cn
(465.89KB)  (450.80KB)  
RNA virus features the high per-generation mutation rate, and thus usually exists as a population presenting intrahost diversity. By the aids of deep sequencing and bioinformatic techniques, recent studies have provided new insights into viral intrahost evolution and viral-host interaction. Currently, many studies have employed the approach of sequencing of viral amplicons (amplicon-seq) to investigate the diversity of viral population. However, there remains concerns about the discrimination of intrahost variations from amplification or sequencing errors, and thus requires assessments on the sensitivity and the specificity of the amplicon-seq approach applying on virus studies. In this study, we designed an assay for assessment of the amplicon-seq approach in the aspects of false negative rate (FNR) and false positive rate (FPR) of intrahost single nucleotide variation (iSNV) calling, accuracy of mutated allele frequency (MuAF) estimation, and MuAF bias between amplicons. We found that when samples had a relatively large viral load (~25 Ct value or ~ 1 × 105 copies/μL), an iSNV calling threshold of MuAF > 0.3% was applicable enables a zero FNR and a <0.001 FPR. The amplicon-seq method could also be applied for challenging samples with a small viral titer (~35 Ct value or ~ 100 copies/μL), and a higher threshold of MuAF > 0.7% could be used to provide comparable FNR and FPR. Therefore, Amplicon-seq, as an efficient and applicable method for clinical and field viral samples, could provide sufficient accuracy and sensitivity for viral intrahost analysis.
Received: 7 May 2018  Accepted: 13 Sep 2018  Published online: 5 Nov 2018
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