Adaptation of single molecule real time (SMRT) sequence technology for hepatitis C virus genome sequencing and identification of resistance-associated substitutions

IF 2.8 3区医学 Q3 VIROLOGY

Virology Pub Date : 2025-03-03 DOI:10.1016/j.virol.2025.110481

Cui Zhang, Pei Liu, Jian Li, Mengjie Han, Yuqiu Liu, Wenge Xing, Maofeng Qiu

{"title":"Adaptation of single molecule real time (SMRT) sequence technology for hepatitis C virus genome sequencing and identification of resistance-associated substitutions","authors":"Cui Zhang, Pei Liu, Jian Li, Mengjie Han, Yuqiu Liu, Wenge Xing, Maofeng Qiu","doi":"10.1016/j.virol.2025.110481","DOIUrl":null,"url":null,"abstract":"<div><h3>Background and objective</h3><div>Hepatitis C virus (HCV) resistance-associated substitutions (RASs) impact HCV treatment with direct-acting antivirals (DAAs). Therefore, a sensitive sequencing assay for detecting HCV RASs is crucial. PacBio sequencing, a Single molecule real time (SMRT) platform, is capable of long-fragment sequencing. This study aims to assess the prospects of PacBio sequencing by comparing its accuracy with Sanger sequencing at distinct thresholds and its cost-effectiveness with next-generation sequencing (NGS).</div></div><div><h3>Methods</h3><div>A total of 28 specimens were selected from the HCV RNA-positive individuals in Linzhou, China. Of these specimens, the NS3 to NS5B regions were amplified and sent for PacBio sequencing. Sequence processing was accomplished by lima, pbmm2 and quasitools software, with threshold setting at 1%, 5%, 10% and 15%.</div></div><div><h3>Results</h3><div>High-frequency RASs (S122G in NS3, R30Q in NS5A, and C316N in NS5B), rare RASs (V55A, R155P, and S174AT in NS3; Y448H in NS5B), and low-threshold RASs (L31Q and L31QFHY in NS5A; L159F in NS5B) were identified. It was found that the results of HCV RASs at the 10% threshold of PacBio sequencing are comparable to those of Sanger sequencing. In terms of high-throughput sequencing, the price of PacBio sequencing (571 CNY per specimen) is significantly lower than that of NGS (1000 CNY per specimen).</div></div><div><h3>Conclusions</h3><div>In summary, these findings suggest that the adoption of the 10% threshold in PacBio sequencing for the analysis of HCV RASs is advisable. Moreover, given its relatively lower cost, PacBio sequencing holds great promise as a valuable tool for large-scale population sequencing.</div></div>","PeriodicalId":23666,"journal":{"name":"Virology","volume":"605 ","pages":"Article 110481"},"PeriodicalIF":2.8000,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Virology","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0042682225000935","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"VIROLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

Background and objective

Hepatitis C virus (HCV) resistance-associated substitutions (RASs) impact HCV treatment with direct-acting antivirals (DAAs). Therefore, a sensitive sequencing assay for detecting HCV RASs is crucial. PacBio sequencing, a Single molecule real time (SMRT) platform, is capable of long-fragment sequencing. This study aims to assess the prospects of PacBio sequencing by comparing its accuracy with Sanger sequencing at distinct thresholds and its cost-effectiveness with next-generation sequencing (NGS).

Methods

A total of 28 specimens were selected from the HCV RNA-positive individuals in Linzhou, China. Of these specimens, the NS3 to NS5B regions were amplified and sent for PacBio sequencing. Sequence processing was accomplished by lima, pbmm2 and quasitools software, with threshold setting at 1%, 5%, 10% and 15%.

Results

High-frequency RASs (S122G in NS3, R30Q in NS5A, and C316N in NS5B), rare RASs (V55A, R155P, and S174AT in NS3; Y448H in NS5B), and low-threshold RASs (L31Q and L31QFHY in NS5A; L159F in NS5B) were identified. It was found that the results of HCV RASs at the 10% threshold of PacBio sequencing are comparable to those of Sanger sequencing. In terms of high-throughput sequencing, the price of PacBio sequencing (571 CNY per specimen) is significantly lower than that of NGS (1000 CNY per specimen).

Conclusions

In summary, these findings suggest that the adoption of the 10% threshold in PacBio sequencing for the analysis of HCV RASs is advisable. Moreover, given its relatively lower cost, PacBio sequencing holds great promise as a valuable tool for large-scale population sequencing.

查看原文本刊更多论文

将单分子实时（SMRT）测序技术用于丙型肝炎病毒基因组测序和耐药性相关替代物的鉴定

背景和目的丙型肝炎病毒（HCV）耐药相关替代药物（ras）影响直接作用抗病毒药物（DAAs）对HCV治疗的影响。因此，一种检测HCV RASs的灵敏测序方法至关重要。PacBio测序是一个单分子实时（SMRT）平台，能够进行长片段测序。本研究旨在通过比较PacBio测序在不同阈值下与Sanger测序的准确性以及与下一代测序（NGS）的成本效益，来评估PacBio测序的前景。方法从林州市HCV rna阳性人群中抽取28份标本。扩增NS3 ~ NS5B区域，进行PacBio测序。序列处理由lima、pbmm2和quasitools软件完成，阈值设置为1%、5%、10%和15%。结果高频RASs (NS3为S122G， NS5A为R30Q， NS5B为C316N)，罕见RASs (NS3为V55A、R155P、S174AT)；低阈值RASs (NS5A中的L31Q和L31QFHY)；NS5B中的L159F)被鉴定。结果发现，PacBio测序在10%阈值下的HCV RASs结果与Sanger测序相当。在高通量测序方面，PacBio测序价格（571元/份）明显低于NGS测序价格（1000元/份）。综上所述，这些研究结果表明，在PacBio测序中采用10%阈值分析HCV RASs是可取的。此外，由于其相对较低的成本，PacBio测序作为大规模种群测序的宝贵工具具有很大的前景。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Virology 医学-病毒学

CiteScore

6.00

自引率

0.00%

发文量

157

审稿时长

50 days

期刊介绍： Launched in 1955, Virology is a broad and inclusive journal that welcomes submissions on all aspects of virology including plant, animal, microbial and human viruses. The journal publishes basic research as well as pre-clinical and clinical studies of vaccines, anti-viral drugs and their development, anti-viral therapies, and computational studies of virus infections. Any submission that is of broad interest to the community of virologists/vaccinologists and reporting scientifically accurate and valuable research will be considered for publication, including negative findings and multidisciplinary work.Virology is open to reviews, research manuscripts, short communication, registered reports as well as follow-up manuscripts.