MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution.
MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution.
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DOI：
10.1093/nar/gkab231
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发表时间：
2021-07-09
影响因子：
14.9
通讯作者：
Torbett BE
中科院分区：
文献类型：
--
作者：
Gallardo CM;Wang S;Montiel-Garcia DJ;Little SJ;Smith DM;Routh AL;Torbett BE
由于RNA病毒具有高度的宿主内多样性，因此在RNA病毒的测序中仍然存在技术挑战。考虑到下一代测序平台的读取长度限制，当询问长距离共同进化的遗传相互作用时，这种瓶颈特别明显。这阻碍了对这些遗传相互作用的直接观察，这些遗传相互作用编码与药物和疫苗开发相关的蛋白质-蛋白质界面。在这里，我们通过开发一种基于纳米孔的远程病毒测序管道来克服这些技术限制，该管道可以从临床样本中获得循环病毒体的准确单分子序列。我们证明了它的效用，在观察个体HIV Gag-Pol基因组的演变，以应对抗病毒的压力。我们的管道，称为多读发夹介导的纠错反应（MrHAMER），以99.9%的准确度产生每个样本> 1000个病毒基因组，保持复杂混合物中存在的测序病毒体的原始比例，并允许检测罕见病毒基因组，其相关突变的频率<1%。该方法有助于对抗病毒治疗和免疫压力的抗性的遗传相关性的可扩展研究，并能够鉴定可调节以获得治疗益处的新型宿主-病毒和病毒-病毒界面。
Technical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolved genetic interactions given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of these genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s of viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure and enables the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.
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影响因子：
4.8
作者：
Waugh C;Cromer D;Grimm A;Chopra A;Mallal S;Davenport M;Mak J
通讯作者：
Mak J
影响因子：
3.7
作者：
Di Giallonardo F;Zagordi O;Duport Y;Leemann C;Joos B;Künzli-Gontarczyk M;Bruggmann R;Beerenwinkel N;Günthard HF;Metzner KJ
通讯作者：
Metzner KJ
DOI：
10.1038/nrg.2017.88
发表时间：
2018-01
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
Gardy JL;Loman NJ
通讯作者：
Loman NJ
影响因子：
48
作者：
Karst, Soren M.;Ziels, Ryan M.;Albertsen, Mads
通讯作者：
Albertsen, Mads
影响因子：
56.9
作者：
Bonhoeffer, S;Chappey, C;Petropoulos, CJ
通讯作者：
Petropoulos, CJ