Single-molecular real-time deep sequencing reveals the dynamics of multidrug resistant haplotypes and structural variations in the hepatitis C virus genome.
Single-molecular real-time deep sequencing reveals the dynamics of multidrug resistant haplotypes and structural variations in the hepatitis C virus genome.
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单分子实时深度测序揭示了丙型肝炎病毒基因组中多重耐药单倍型的动态和结构变异。
DOI：
10.1038/s41598-020-59397-2
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Sekine A
中科院分区：
文献类型：
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作者：
Yamashita T;Takeda H;Takai A;Arasawa S;Nakamura F;Mashimo Y;Hozan M;Ohtsuru S;Seno H,Ueda Y;Sekine A
虽然丙型肝炎病毒（HCV）的直接作用抗病毒药物（DAA）已经取得了显着进展，但患者仍然遭受治疗失败。为了彻底根除HCV，在单克隆水平上对多耐药相关取代（RAS）有更深入的了解是必不可少的。为了了解HCV准种及其在DAA治疗期间的动态，我们对12名DAA治疗失败的基因型1b型HCV感染患者的血清进行了单分子实时（SMRT）深度测序，包括治疗前和治疗后。我们总共鉴定了187，539个克隆的NS3和NS5A基因之间> 3.2kbp的序列，基于7个RAS基因座的连锁将其分类为单倍型代码。在处理期间，每个样品的单倍型编码的数量从14.67 ± 9.12显著降低到6.58 ± 7.1，而每个克隆的7个RAS基因座上的非同义密码子的数量从1.50 ± 0.92显著增加到3.64 ± 0.75。在5例病例中，治疗前的少数多药耐药单倍型与复发时的主要单倍型相同。此外，各种结构的变化（SV）进行了检测和动态分析。这些结果表明，SMRT深度测序可用于检测少数单倍型和SV，并在单克隆水平上评估病毒基因组的动态。
While direct-acting antivirals (DAAs) for hepatitis C virus (HCV) have dramatically progressed, patients still suffer from treatment failures. For the radical eradication of HCV, a deeper understanding of multiple resistance-associated substitutions (RASs) at the single-clone level is essential. To understand HCVquasispeciesand their dynamics during DAA treatment, we applied single-molecule real-time (SMRT) deep sequencing on sera from 12 patients with genotype-1b HCV infections with DAA treatment failures, both pre- and post-treatment. We identified >3.2 kbp sequences between NS3 and NS5A genes of 187,539 clones in total, classifying into haplotype codes based on the linkage of seven RAS loci. The number of haplotype codes during the treatment, per sample, significantly decreased from 14.67 ± 9.12 to 6.58 ± 7.1, while the number of nonsynonymous codons on the seven RAS loci, per clone, significantly increased from 1.50 ± 0.92 to 3.64 ± 0.75. In five cases, the minority multi-drug resistant haplotypes at pre-treatment were identical to the major haplotypes at relapse. Moreover, various structural variations (SVs) were detected and their dynamics analysed. These results suggest that SMRT deep sequencing is useful for detecting minority haplotypes and SVs, and to evaluate the dynamics of viral genomes at the single-clone level.