Ultrasensitive Single-Genome Sequencing to Study HIV Transmission and Evolution

背景：我们在16年前开发和发表的单基因组测序测定（SGS）（Palmer et al.，J. Clin. Microbiol. 43：406-413，2005）仍然是病毒群体遗传分析的金标准。尽管下一代测序（NGS）为更深入地研究病毒种群提供了潜力，但文库构建期间的PCR错误、偏差和重组限制了其在种群测序和非连锁等位基因频率测量中的应用。在该项目中，我们先前开发了一种用于NGS文库构建的新方法，该方法减少了PCR偏倚和错误，从最终数据集中消除了PCR重组体，并产生了数千个与SGS质量相同但变体多100倍的单基因组序列（Boltz et al. Retrovirology 13：87，2016）。这种新的超灵敏SGS（uSGS）检测方法不仅用于检测罕见等位基因的连锁，包括耐药突变，还用于病毒群体的深入系统发育分析。_NGS的标准方法无法再现SGS最重要的特性，这实际上消除了PCR伪影，但受到可以获得的序列数量有限的限制（Palmer等人，J. Clin. Microbiol. 43：406-413，2005）。为了解决这些问题中的一些，将引物ID（包含4-10个随机核苷酸的分子标签）掺入cDNA合成引物中，使得通过逆转录产生的每个cDNA分子被独特地标记（Jabara等人，PNAS 108：20166-20171，2011）。然后通过PCR扩增引物ID标记的cDNA，并通过NGS对子代扩增子进行测序。接下来，序列读数通过其共同引物ID分箱，揭示PCR模板反应。分箱序列的比对有助于鉴定PCR错误和PCR重组，使得可以从每个箱中的比对产生一个共有序列。尽管使用引物ID可能会导致引物ID本身出现PCR错误，但可以使用过滤技术来检测和排除存在PCR错误的引物ID（Zhou等人，J. Virol. 89：8540-8555，2015）。因此，引物ID在鉴定NGS文库生成期间引入的错误方面非常有效，并且提供了可以使用PCR和NGS准确检测和测量HIV RNA群体中罕见等位基因频率的唯一手段。_对于文库生成，NGS需要连接衔接子序列，以在Illumina流动池中进行文库捕获、扩增和测序。一种连接这些衔接子的现有方法采用含有长的5 '末端延伸的PCR引物。我们和其他人表明，这种“长引物PCR”方法（LP-PCR）产生高水平的PCR重组以及低效和不均匀的扩增（Jabara等人，PNAS 108：20166-20171，2011; Shao等人，Retrovirology 10：18，2013）。重组序列构成了数据的很大一部分，使得最终结果对于鉴定罕见的单倍型或进行准确的系统发育分析是不可靠的。因此，我们开发了一种用于NGS文库构建的新方法，该方法扩增更高比例的cDNA分子，同时显著降低PCR偏倚和重组。PCR错误和体外重组体通过一种新的分析管道被检测和去除，从而产生的序列数据可以与SGS检测的准确性和可靠性相媲美，但测序深度要高出100倍。我们的方法结合了有限循环PCR和高效的衔接子连接方法。我们称这种方法为超灵敏SGS（uSGS）。_完成：由于uSGS检测试剂盒从数万个HIV模板中生成数百万个测序读段，因此无法使用标准的序列分析方法（例如，检测耐药性突变）。标准方法只能处理和注释多达100个HIV基因组。为了克服这一障碍，我们开发了一种新的生物信息学工具，该工具可以检测和报告从临床样品获得的数十万个HIV基因组的数据集中HIV耐药性突变的联系（Shao et al.，AIDS Res. Retroviruses 36：942-947，2020）。使用这个工具，我们证明了HIV耐药突变有时在ART开始之前与相同的病毒基因组相关联，并且这种联系是ART失败的重要原因。_使用我们的SGS检测，我们证明了当病毒变体的供体群体中存在耐药性突变时，暴露前预防可能无法预防HIV传播（Spinelli et al.，临床感染72：2025-2028，2021）。_与Eric Freed博士（HIV动力学和复制计划）的实验室合作，我们研究了脱靶突变在ART未能控制体内HIV复制中的作用。我们鉴定了HIV env基因的gp 120-gp 41界面中的几个突变，其能够赋予对多种ARV的低至中等水平抗性（Hikichi等人，mBio12：e03134-20，2021）。

BACKGROUND: The single-genome sequencing assay (SGS) that we developed and published 16 years ago (Palmer et al., J. Clin. Microbiol. 43:406-413, 2005) remains the gold standard for genetic analysis of viral populations. Although next-generation sequencing (NGS) offers the potential for studying virus populations in much greater depth, PCR error, bias, and recombination during library construction have limited its use to population sequencing and measurements of unlinked allele frequencies. In this project, we previously developed a new method for NGS library construction that reduces PCR bias and error, eliminates PCR recombinants from the final datasets, and generates thousands of single-genome sequences of the same quality as SGS but with 100-fold more variants (Boltz et al., Retrovirology 13:87, 2016). This new, ultrasensitive SGS (uSGS) assay has been used not only for detecting linkage of rare alleles, including drug-resistance mutations, but also for in-depth phylogenetic analyses of viral populations. ____Standard methods for NGS fall short of reproducing the most important properties of SGS, which virtually eliminates PCR artifacts but is constrained by the limited number of sequences that can be obtained (Palmer et al., J. Clin. Microbiol. 43:406-413, 2005). To address some of these issues, primer IDs (molecular tags comprising 4-10 random nucleotides) are incorporated into cDNA synthesis primers so that each cDNA molecule generated by reverse transcription is uniquely labeled (Jabara et al., PNAS 108:20166-20171, 2011). Primer ID-tagged cDNAs are then amplified by PCR and daughter amplicons are sequenced by NGS. Next, sequence reads are binned by their common primer ID, revealing PCR template resampling. Alignment of binned sequences facilitates identification of PCR errors and PCR recombination such that one consensus sequence can be generated from the alignments in each bin. Although the use of primer IDs can result in PCR errors within the primer ID itself, filtering techniques can be used to detect and exclude primer IDs with PCR errors (Zhou et al., J. Virol. 89:8540-8555, 2015). As such, primer IDs are extremely effective in identifying errors introduced during NGS library generation and provide the only means by which rare allele frequencies in HIV RNA populations may be accurately detected and measured using PCR and NGS. ____For library generation, NGS requires the attachment of adaptor sequences for library capture, amplification, and sequencing in the Illumina flow cell. One current method to attach these adaptors employs PCR primers containing lengthy 5'-terminal extensions. We and others showed that this "long primer PCR" method (LP-PCR) produces high levels of PCR recombination as well as inefficient and nonuniform amplification (Jabara et al., PNAS 108:20166-20171, 2011; Shao et al., Retrovirology 10:18, 2013). Recombinant sequences constitute a large fraction of the data, making the final results unreliable for identifying rare haplotypes or for performing accurate phylogenetic analysis. Accordingly, we developed a new method for NGS library construction that amplifies a higher fraction of cDNA molecules with significantly reduced PCR bias and recombination. PCR errors and in vitro recombinants are detected and removed through a novel analysis pipeline, resulting in sequence data that rival the accuracy and reliability of the SGS assay but with 100-fold greater sequencing depth. Our method combines limited-cycle PCR with a highly efficient method of adaptor ligation. We call the approach ultrasensitive SGS (uSGS). ____ACCOMPLISHMENTS: Because the uSGS assay generates millions of sequencing reads from tens of thousands of HIV templates, standard methods for sequence analysis (for example, to detect drug-resistance mutations) cannot be used. The standard methods only have the ability to process and annotate up to 100 HIV genomes. To overcome this obstacle, we developed a new bioinformatic tool that can detect and report the linkage of HIV drug-resistance mutations in datasets of hundreds of thousands of HIV genomes obtained from clinical samples (Shao et al., AIDS Res. Hum. Retroviruses 36:942-947, 2020). Using this tool, we demonstrated that HIV drug-resistance mutations are, on occasion, linked on the same viral genomes prior to the initiation of ART and that this linkage is an important cause of ART failure. ___Using our SGS assays, we demonstrated that pre-exposure prophylaxis can fail to prevent HIV transmission when drug-resistance mutations are present in the donor population of viral variants (Spinelli et al., Clin. Infect. Dis. 72:2025-2028, 2021). ___In collaboration with the lab of Dr. Eric Freed (HIV Dynamics and Replication Program), we investigated the role of off-target mutations in the failure of ART to control HIV replication in vivo. We identified several mutations in the gp120-gp41 interface of the HIV env gene that are capable of conferring low- to medium-level resistance to multiple ARVs (Hikichi et al., mBio12:e03134-20, 2021).