Ultrasensitive Single-Genome Sequencing to Study HIV Transmission and Evolution

背景：我们在16年前开发并发表的单基因组测序测定（SGS） (Palmer et al.， J. clint .；微生物学。43:406-413,2005)仍然是病毒种群遗传分析的黄金标准。虽然下一代测序（NGS）提供了更深入研究病毒群体的潜力，但在文库构建过程中，PCR错误、偏差和重组限制了其在群体测序和非连锁等位基因频率测量中的应用。在这个项目中，我们之前开发了一种新的NGS文库构建方法，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，并生成了数千个与SGS相同质量的单基因组序列，但变体多了100倍（Boltz等人，Retrovirology 13:87, 2016）。这种新的超灵敏的SGS （uSGS）检测不仅用于检测罕见等位基因的连锁，包括耐药突变，而且还用于病毒种群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。微生物学杂志。43:406-413,2005)。为了解决其中的一些问题，引物id（包含4-10个随机核苷酸的分子标签）被整合到cDNA合成引物中，以便通过反转录生成的每个cDNA分子都被唯一标记（Jabara等人，PNAS 108:20166-20171, 2011）。引物id标记的cdna然后用PCR扩增，子扩增子用NGS测序。接下来，序列读取由它们的共同引物ID分类，揭示PCR模板重采样。分仓序列比对有助于鉴定PCR错误和PCR重组，从而可以从每个分仓的比对中产生一个一致的序列。虽然使用引物ID会导致引物ID本身的PCR错误，但过滤技术可以用来检测和排除PCR错误的引物ID （Zhou et al.， J. Virol. 89:8540-8555, 2015）。因此，引物id在识别NGS文库生成过程中引入的错误方面非常有效，并提供了使用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需要连接适配器序列，以便在Illumina流动细胞中进行文库捕获，扩增和测序。目前连接这些接头的一种方法是使用含有长5'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，使得最终结果在识别罕见的单倍型或进行准确的系统发育分析时不可靠。因此，我们开发了一种新的NGS文库构建方法，该方法可以扩增更高比例的cDNA分子，显著降低PCR偏倚和重组。PCR错误和体外重组通过一种新的分析管道进行检测和去除，从而产生序列数据，其准确性和可靠性可与SGS检测相媲美，但测序深度要高100倍。我们的方法结合了有限循环PCR和高效接头连接方法。我们称这种方法为超灵敏SGS （uSGS）。____取得的成就：由于美国地质勘探局的测定从数以万计的HIV模板中产生数百万个测序读数，因此无法使用序列分析的标准方法（例如，检测耐药突变）。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，可以在从临床样本中获得的数十万HIV基因组数据集中检测和报告HIV耐药突变的联系（Shao等人，AIDS Res. Hum.）。逆转录病毒36:942-947,2020)。使用这个工具，我们证明了HIV耐药突变，有时，在开始抗逆转录病毒治疗之前，在相同的病毒基因组上连接，这种连接是抗逆转录病毒治疗失败的一个重要原因。使用我们的SGS检测，我们证明，当病毒变体的供体人群中存在耐药突变时，暴露前预防可能无法防止HIV传播（Spinelli等人，临床）。感染。Dis. 72:2025- 2028,2021)。我们与Eric Freed博士（HIV Dynamics and Replication Program）的实验室合作，研究了脱靶突变在ART无法控制HIV在体内复制中的作用。我们在HIV env基因的gp120-gp41界面上发现了几个突变，这些突变能够赋予对多种抗逆转录病毒药物低至中等水平的耐药性（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).