Ultrasensitive Single-Genome Sequencing to Study HIV Transmission and Evolution

背景：我们在11年前开发并发表了单基因组测序测定（SGS，有时称为SGA）。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，我们正在开发一种新的NGS文库构建方法，该方法可以减少PCR偏倚和错误，从最终数据集中消除PCR重组，并生成数千个与SGS质量相同但变体多100倍的单基因组序列。这种新的超灵敏的SGS （uSGS）检测方法不仅将用于检测罕见等位基因的连锁，包括耐药突变，而且还将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. clinin。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（Shao等人，Retrovirology 10:18, 2013； Jabara等人，PNAS 108:20166-20171, 2011）。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，我们正在开发一种新的NGS文库构建方法，该方法可以扩增更高比例的cDNA分子，显著减少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。我们称这种新方法为超灵敏SGS （uSGS）。当我们将这种新方法与传统的LP-PCR方法进行目标NGS文库生成的比较时，我们发现LP-PCR方法不仅由于非常高的体外重组率而无法检测罕见变异之间的连锁，而且uSGS方法可以纠正这一缺陷。到目前为止，我们完成的实验表明，uSGS的结果是更完整的cDNA文库采样，以及更严格的序列过滤，提供了几乎没有PCR错误和PCR重组的数据集。因此，我们的新uSGS方法是迄今为止研究HIV-1群体结构和进化以及检测罕见等位基因之间联系的最有效手段。我们计划应用我们的新uSGS来完成我们对HIV耐药突变出现的研究，其中我们正在调查有和没有事先暴露于单剂量奈韦拉平（sdNVP）的妇女中低频突变的联系。体内HIV-1感染的高度遗传多样性的一个后果是，在开始抗逆转录病毒治疗（ART）之前，复制群体中存在耐药性突变。我们过去的研究包括调查这些预先存在的耐药突变在首次治疗患者和rt - shiv感染猕猴的血浆中出现的频率，确定它们对后续治疗的影响，以及确定HIV-1多样性对治疗结果的总体影响(Boltz等人，PNAS 108:9202-9207, 2011； Boltz等人，J. Virol. 86: 1225 -12530, 2012； Boltz等人，J.感染。疾病。209:703-710,2014)。例如，我们调查了之前接触过或没有接触过snvp的女性中低频nvp耐药突变对随后联合ART的影响（A5208试验1和试验2）。我们发现，与未检测到NVP耐药性的女性相比，在联合抗逆转录病毒治疗前曾暴露于snvp且携带NVP抗性突变频率高于1.0%的女性在给予含NVP的抗逆转录病毒治疗时出现病毒学失败的风险明显更高。然而，未接受sdNVP且先前存在低频nvp耐药突变的妇女没有增加病毒学失败的风险，尽管这些突变有时在血浆中出现的频率与接受sdNVP和抗逆转录病毒治疗失败的妇女相同。我们假设，在先前暴露于sdNVP的妇女中，病毒学失败与先前存在的低频nvp耐药突变的关联是由于nvp耐药突变与对联合抗逆转录病毒治疗中使用的其他类别抑制剂产生耐药的突变的联系。耐药突变的连锁反应可能发生在这些妇女接受snvp后不久，当时nvp耐药突变的频率要高得多（高达病毒总数的80%）。nvp耐药变异的高频率可能使这些突变与人群中随机出现的其他预先存在的耐药突变联系起来，最终导致后续联合抗逆转录病毒治疗失败。相比之下，nvp耐药突变很可能从未在未暴露于sdNVP的妇女中出现高频率，因此不太可能与其他先前存在的耐药突变联系起来。因此，抗逆转录病毒疗法能够在随机出现nvp耐药突变的女性中实现持续抑制，而不是在snvvp的选择性压力下。为了验证这一假设，我们正在开发一种检测方法，使我们能够调查血浆中低频耐药突变的联系，并计划将该检测方法用于A5208妇女的抗逆转录病毒治疗前样本，以确定其联系谱。我们将应用新的uSGS方法来完成这些最终的研究，以调查有或没有先前暴露于snvp的女性中低频突变的联系。在完成这项试验后，我们将利用美国地质勘探局来解决与艾滋病毒的传播、进化和储存有关的问题。

BACKGROUND: The single-genome sequencing assay (SGS, sometimes called SGA) that we developed and published 11 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 are developing 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. This new, ultrasensitive SGS (uSGS) assay will be 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 non-uniform amplification (Shao et al., Retrovirology 10:18, 2013; Jabara et al., PNAS 108:20166-20171, 2011).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 are developing 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 new approach ultrasensitive SGS (uSGS). ACCOMPLISHMENTS: When we compared this new approach to conventional LP-PCR for targeted NGS library generation, we found that not only is detection of linkage among rare variants not possible using LP-PCR due to very high in vitro recombination rates, but also that the uSGS method can correct this deficiency. Our experiments completed thus far show that uSGS results in more complete sampling of cDNA libraries and, together with more stringent filtering of the sequences, provides datasets that are nearly free of PCR error and PCR recombination. Consequently, our novel uSGS methodology is the most effective means developed to date for studying HIV-1 population structure and evolution as well as for detecting linkage among rare alleles.We plan to apply our new uSGS to complete our studies on the emergence of HIV drug-resistance mutations in which we are investigating the linkage of low-frequency mutations in women with and without prior exposure to a single dose of nevirapine (sdNVP). A consequence of the high genetic diversity of HIV-1 infection in vivo is the existence of drug-resistance mutations in the replicating population before the initiation of antiretroviral therapy (ART).Our past studies included investigating the frequency with which these preexisting drug-resistance mutations are present in the plasma of treatment-naive patients and RT-SHIV-infected macaques, determining their effect on subsequent therapy, and determining the overall impact of HIV-1 diversity on treatment outcome (Boltz et al., PNAS 108:9202-9207, 2011; Boltz et al., J. Virol. 86:12525-12530, 2012; Boltz et al., J. Infect. Dis. 209:703-710, 2014). For example, we investigated the effect of low-frequency NVP-resistance mutations in women who had and did not have previous exposure to sdNVP on subsequent combination ART (A5208 Trial 1 and Trial 2). We found that women who had prior exposure to sdNVP and carried NVP-resistance mutations at a frequency above 1.0% before combination ART were at significantly higher risk of virologic failure when given NVP-containing ART than women without detectable NVP resistance. However, women who did not receive sdNVP and had low-frequency preexisting NVP-resistance mutations were not at increased risk for virologic failure, despite these mutations sometimes being present in the plasma in the same frequencies as women who had received sdNVP and failed ART. We hypothesized that the association of virologic failure with preexisting, low-frequency NVP-resistance mutations in women who had prior exposure to sdNVP was due to linkage of NVP-resistance mutations to mutations that confer resistance to other classes of inhibitors used in the combination ART. Linkage of drug-resistance mutations likely occurred in these women shortly after receiving sdNVP when the frequencies of NVP-resistance mutations were at much higher levels (up to 80% of the total virus population). The high frequency of NVP-resistant variants likely allowed for these mutations to become linked to other preexisting drug-resistance mutations in the population that arose stochastically, ultimately resulting in failure of subsequent combination ART. By contrast, NVP-resistance mutations were most likely never present in high frequencies in women who had not been exposed to sdNVP and, therefore, did not likely become linked to other preexisting drug-resistance mutations. As a result, ART was able to achieve sustained suppression in women with NVP-resistance mutations that arose stochastically rather than under the selective pressure of sdNVP. To test this hypothesis, we are developing an assay that will allow us to investigate the linkage of low-frequency drug-resistance mutations in the plasma and plan to use this assay on pre-ART samples from women in A5208 to determine their linkage profiles. We will apply our new uSGS method to complete these final studies on investigating the linkage of low-frequency mutations in women with and without prior exposure to sdNVP. After completing this trial, we will use uSGS to address questions related to the transmission, evolution, and reservoir of HIV.