Ultrasensitive Single-Genome Sequencing to Study HIV Transmission and Evolution

Division Of Basic Sciences - Nci

Investigators

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Abstract

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).