Reference Plasmid pHXB2_D is an HIV-1 Molecular Clone that Exhibits Identical LTRs and a Single Integration Site Indicative of an HIV Provirus

Objective: To compare long-read nanopore DNA sequencing (DNA-seq) with short-read sequencing-by-synthesis for sequencing a full-length (e.g., non-deletion, nor reporter) HIV-1 model provirus in plasmid pHXB2_D. Design: We sequenced pHXB2_D with long- and short-read DNA-seq, evaluating sample variability with resequencing (sequencing and mapping to reference HXB2) and de novo viral genome assembly. Methods: We prepared pHXB2_D for long-read nanopore DNA-seq, varying DNA polymerases Taq (Sigma-Aldrich) and Long Amplicon (LA) Taq (Takara). Nanopore basecallers were compared. After aligning reads to the reference HXB2 to evaluate sample coverage, we looked for variants. We next assembled reads into contigs, followed by finishing and polishing. We hired an external core to sequence-verify pHXB2_D with single-end 150 base-long Illumina reads, after masking sample identity. Results: We achieved full-coverage (100%) of HXB2 HIV-1 from 59 to 39 long terminal repeats (LTRs), with median per-base coverage of over 9000x in one experiment on a single MinION flow cell. The longest HIV-spanning read to-date was generated, at a length of 11,487 bases, which included full-length HIV-1 and plasmid backbone with flanking host sequences supporting a single HXB2 integration event. We discovered 20 single nucleotide variants in pHXB2_D compared to reference, verified by short-read DNA sequencing. Conclusions: Nanopore sequencing performed as-expected, even covering full-length HIV. The discovery of variants in a reference plasmid demonstrates the need for sequence verification moving forward, in line with calls from funding agencies for reagent verification. These results illustrate the utility of long-read DNA-seq to advance the study of HIV at single integration site resolution.