title: Ultrasensitive single-genome sequencing: accurate, targeted, next generation sequencing of HIV-1 RNA.
abstract: BACKGROUND
  Although 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.
  
  
  RESULTS
  Primer 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: HIV Dynamics and Replication Program, CCR, National Cancer Institute, NIH, Translational Research Unit, 105 Boyles Street, Building 535 Room 111, Frederick, MD, 21702-1201, USA. boltzv@mail.nih.gov.
authors.forename: Valerie F
authors.initials: VF
authors.lastname: Boltz
authors.affiliation: HIV Dynamics and Replication Program, CCR, National Cancer Institute, NIH, Translational Research Unit, 105 Boyles Street, Building 535 Room 111, Frederick, MD, 21702-1201, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Rausch
authors.affiliation: Frederick National Laboratory for Cancer Research, Advanced Biomedical Computing Center, Leidos Biomedical Research, Inc, Frederick, MD, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Shao
authors.affiliation: HIV Dynamics and Replication Program, CCR, National Cancer Institute, NIH, Translational Research Unit, 105 Boyles Street, Building 535 Room 111, Frederick, MD, 21702-1201, USA.
authors.forename: Junko
authors.initials: J
authors.lastname: Hattori
authors.affiliation: Frederick National Laboratory for Cancer Research, Advanced Biomedical Computing Center, Leidos Biomedical Research, Inc, Frederick, MD, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Luke
authors.affiliation: HIV Dynamics and Replication Program, CCR, National Cancer Institute, NIH, Translational Research Unit, 105 Boyles Street, Building 535 Room 111, Frederick, MD, 21702-1201, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Maldarelli
authors.affiliation: Division of Infectious Disease, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Mellors
authors.affiliation: HIV Dynamics and Replication Program, CCR, National Cancer Institute, NIH, Translational Research Unit, 105 Boyles Street, Building 535 Room 111, Frederick, MD, 21702-1201, USA.
authors.forename: Mary F
authors.initials: MF
authors.lastname: Kearney
authors.affiliation: Department of Molecular Biology and Microbiology, Tufts University, Boston, MA, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Coffin
mesh_terms: D002874:Chromosome Mapping; D017931:DNA Primers; D000068598:Data Accuracy; D024882:Drug Resistance, Viral; D015723:Gene Library; D016679:Genome, Viral; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral
keywords: Allele linkage; Deep sequencing; HIV; HIV drug resistance; Minority variants; NGS; Primer ID; SGS; Single-genome sequencing; Supermajority correction; Targeted next-generation sequencing