Ultrasensitive single-genome sequencing: accurate, targeted, next generation sequencing of HIV-1 RNA

Abstract

BackgroundAlthough next generation sequencing (NGS) offers the potential for studying virus populations in unprecedented depth, PCR error, amplification bias and recombination during library construction have limited its use to population sequencing and measurements of unlinked allele frequencies. Here we report a method, termed ultrasensitive Single-Genome Sequencing (uSGS), for NGS library construction and analysis that eliminates PCR errors and recombinants, and generates single-genome sequences of the same quality as the “gold-standard” of HIV-1 single-genome sequencing assay but with more than 100-fold greater depth.ResultsPrimer ID tagged cDNA was synthesized from mixtures of cloned BH10 wild-type and mutant HIV-1 transcripts containing ten drug resistance mutations. First, the resultant cDNA was divided and NGS libraries were generated in parallel using two methods: uSGS and a method applying long PCR primers to attach the NGS adaptors (LP-PCR-1). Second, cDNA was divided and NGS libraries were generated in parallel comparing 3 methods: uSGS and 2 methods adapted from more recent reports using variations of the long PCR primers to attach the adaptors (LP-PCR-2 and LP-PCR-3). Consistently, the uSGS method amplified a greater proportion of cDNAs, averaging 30% compared to 13% for LP-PCR-1, 21% for LP-PCR-2 and 14% for LP-PCR-3. Most importantly, when the uSGS sequences were binned according to their primer IDs, 94% of the bins did not contain PCR recombinant sequences versus only 55, 75 and 65% for LP-PCR-1, 2 and 3, respectively. Finally, when uSGS was applied to plasma samples from HIV-1 infected donors, both frequent and rare variants were detected in each sample and neighbor-joining trees revealed clusters of genomes driven by the linkage of these mutations, showing the lack of PCR recombinants in the datasets.ConclusionsThe uSGS assay can be used for accurate detection of rare variants and for identifying linkage of rare alleles associated with HIV-1 drug resistance. In addition, the method allows accurate in-depth analyses of the complex genetic relationships of viral populations in vivo.

HIV-DRLink: A tool for detecting linked HIV-1 drug resistance mutations in next generation sequencing data

“…To evaluate true linkage of drug resistance mutations on single HIV-1 genomes, we tested the pipeline on plasma HIV-1 RNA sequences obtained by uSGS (Boltz et al, 2016) (sequences available at KY810858-KY812454). The plasma sample was obtained from an HIV-infected donor with viremic failure on ART.…”

“…The plasma sample was obtained from an HIV-infected donor with viremic failure on ART. uSGS yielded 1597 high quality single genome pol sequences covering RT codons 59 to 131 and 166 to 226 (Boltz et al, 2016). Table 2 shows 12 different resistance patterns that were detected using the HIV-DRLink program, all of which had linked mutations.…”

“…While HIV-DRLink can be used to detect linkage of drug resistance mutations in sequences obtained by technologies that omit artifactual recombination, Illumina MiSeq-based uSGS data was used here to validate the pipeline on sequences obtained from a clinical sample (Boltz et al, 2016). The clinically-derived sequences were obtained from genbank (accession #’s KY810858-KY812454).…”

“…The clinically-derived sequences were obtained from genbank (accession #’s KY810858-KY812454). After filtering to remove low quality reads, the paired end fastq files were used for bioinformatics processing to generate HIV-1 sequences of 404 bases in length that covered RT from codons 59 to 131 and 166 to 226 (Boltz et al, 2016).…”

“…One difficulty in the interpretation of linked mutations is that bulk PCR may result in artifactual recombination (Shao et al, 2013). Therefore, detection of linked mutations can only be applied to sequences obtained using methods that reduce PCR recombination in conjunction with pipelines that omit sequences resulting from PCR recombination, such as Single-Genome Sequencing (SGS) (Palmer et al, 2005), ultrasensitive Single-Genome Sequencing (uSGS) (Boltz et al, 2016), or other similar next-generation sequencing (NGS) methods (Jabara et al, 2011; Zhou et al, 2015). In brief, uSGS uses primer IDs like many other NGS approaches (Casbon et al, 2011; Fu et al, 2011; Kinde et al, 2011; Liang et al, 2014), however, the Illumina adapters are added by ligation rather than by PCR, significantly reducing the bias and recombination that is inherent to amplification with long PCR primers.…”