Deep Sequencing of HIV Viral Genomes

Deep HIV viral sequencing using a nested PCR approach was performed on subject plasma samples at IIID, Murdoch University (Perth, WA, Australia), as previously reported [13 (link)]. Briefly, HIV RNA was extracted from subject plasma samples using the Life Technologies MagMAX-96 Viral RNA Isolation Kit, as per the manufacturer’s instructions. Targeted RT-PCRs were performed to cover the Gag, Pol and Nef genes. Resultant first round products were amplified by nested PCR. Deep bulk sequencing was conducted on the Illumina MiSeq platform. Second round PCR amplicons were quantified, equimolar pooled and enzymatically fragmented for each individual. Raw sequencing reads were quality trimmed (default MiSeq settings) and aligned to the HXB2 reference HIV clade B strain (GenBank accession number K03455) using QIAGEN Bioinformatics’ CLCbio Genomics Workbench 11. Aligned sequencing files were exported in BAM format and imported into an in-house developed analysis tool (http://www.iiid.com.au/software/vgas), with amino acid frequencies, consensus and majority sequences exported using a 3% nucleotide cut off.