An Oxford Nanopore Technology-Based Hepatitis B Virus Sequencing Protocol Suitable For Genomic Surveillance Within Clinical Diagnostic Settings.
An Oxford Nanopore Technology-Based Hepatitis B Virus Sequencing Protocol Suitable For Genomic Surveillance Within Clinical Diagnostic Settings.
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基于牛津纳米孔技术的乙型肝炎病毒测序方案，适用于临床诊断环境中的基因组监测。
DOI：
10.1101/2024.01.19.24301519
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Sanko,TomaszJanu
中科院分区：
文献类型：
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作者：
Tshiabuila,Derek;Choga,Wonderful;James,SanE;Maponga,Tongai;Preiser,Wolfgang;vanZyl,Gert;Moir,Monika;vanWyk,Stephanie;Giandhari,Jennifer;Pillay,Sureshnee;Anyaneji,UgochukwuJ;Lessells,RichardJ;Naidoo,Yeshnee;Sanko,TomaszJanu
慢性乙型肝炎病毒（HBV）感染仍然是一个重大的公共卫生问题，特别是在负担沉重的非洲。HBV是一种包膜病毒，可分为十种不同的基因型（A-J）。确定HBV基因型的测试是基于全基因组测序或反向杂交。在实践中，这两种方法都有局限性。然而，通常使用Sanger方法的诊断性测序往往只关注s基因，很少或根本没有关于患者内部HBV遗传多样性的信息，而反向杂交仅检测已知的基因型特异性突变。为了解决这些限制，我们开发了一种基于牛津纳米孔技术（ONT）的HBV诊断测序方案，适用于临床病毒学，可产生完整的基因组序列和广泛的患者内HBV多样性数据。具体而言，该方案包括基于平片的HBV序列PCR扩增，使用ONT快速条形码试剂盒（Oxford nanopore Technologies, Oxford, OX4 4DQ， UK）制备文库，使用ONT GridION测序，使用基因组检测软件v1.132/1.133进行基因分型，使用jpHMM（2011年10月26日版本）和RDP5.61软件进行重组分析，以及使用Geno2pheno v2.0软件进行耐药性分析。我们通过从南非西开普省HBV感染患者的148个剩余诊断样本中高效地生成和表征高质量的近全长HBV基因组，证明了我们方案的实用性，为世界该地区HBV的遗传多样性和流行病学提供了有价值的见解。
Chronic Hepatitis B Virus (HBV) infection remains a significant public health concern, particularly in Africa, where the burden is substantial. HBV is an enveloped virus, classified into ten phylogenetically distinct genotypes (A–J). Tests to determine HBV genotypes are based on full-genome sequencing or reverse hybridization. In practice, both approaches have limitations. Whereas diagnostic sequencing, generally using the Sanger approach, tends to focus only on the S-gene and yields little or no information on intra-patient HBV genetic diversity, reverse hybridization detects only known genotype-specific mutations. To resolve these limitations, we developed an Oxford Nanopore Technology (ONT)-based HBV diagnostic sequencing protocol suitable for clinical virology that yields both complete genome sequences and extensive intra-patient HBV diversity data. Specifically, the protocol involves tiling-based PCR amplification of HBV sequences, library preparation using the ONT Rapid Barcoding Kit (Oxford nanopore Technologies, Oxford, OX4 4DQ, UK), ONT GridION sequencing, genotyping using genome detective software v1.132/1.133, a recombination analysis using jpHMM (26 October 2011 version) and RDP5.61 software, and drug resistance profiling using Geno2pheno v2.0 software. We prove the utility of our protocol by efficiently generating and characterizing high-quality near full-length HBV genomes from 148 residual diagnostic samples from HBV-infected patients in the Western Cape province of South Africa, providing valuable insights into the genetic diversity and epidemiology of HBV in this region of the world.