Sis approach would be required to use this method in a
Sis approach would be required to use this method in a clinical setting, however, the provided methods are relatively quick and simple for research-based projects and is considerably faster than Sanger-based data analysis of 64 samples. One of the sequence analysis challenges with working with HIV drug resistance is that resistance is defined by changes in amino acid sequence that could be rendered by several different nucleotide variants. Many analysis tools consider a single nucleotide variant at a time and not necessarily the effect of two nucleotide changes from a single sequence within a codon that might change an amino acid. For example, a variant caller might call a change from ATG (Met) to GTA (Val) as a mix of ATA (Ile) and GTG (Val), considering each single nucleotide change away from ATG as separate even though they are present in the same sequence. We have worked with the developers at Geneious to create a phased variant detection approach that is accurate when assessing nucleotide variants in the context of amino acid changes, which is paramount to our analysis approach. This variant detection approach is now built into the latest Geneious software release. Lastly, while we can identify drug resistance mutations in the protease, reverse transcriptase PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27107493 and integrase regions simultaneously with this assay, given the nature of library formation and Illumina MiSeq sequencing we cannot necessarily associate together mutations found in one area of the pol gene product with mutations found in another area. With the paired-end technology it is possible to link one mutation from the same template to a mutation found 600 bp downstream, but we cannot make those associations with mutations that span further apart. In this regard, this assay is not more informative than traditional genotyping assays using Sanger-based sequencing, which also cannot associate mutations together across multiple pol gene products. The only way to reliably span the entire pol gene from a single virus involves either cloning and sequencing or limiting dilution PCR and sequencing. However, sequencing technologies continue to improve and as error rates improve for techniques that sequence thousands of TAPI-2 site nucleotides in a single sequencing read, linking together all drug resistance mutations will become PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28242652 much easier.Dudley et al. Retrovirology (Page 11 ofConclusions We have designed a universal primer set that amplifies part of the pol gene from the major HIV-1 subtypes A, B, C, D, CRF01_AE and CRF02_AG. These primers were used in an Illumina MiSeq-based sequencing method for the detection of drug resistance in primary HIV samples from individuals failing treatment after release from prison. We characterized drug resistance to protease, reverse transcriptase and integrase inhibitors simultaneously. 54 of the patients failing treatment harbored mutations in their virus at the time of treatment failure that were not present before treatment. Of the patients with virus harboring major drug resistance mutations, all but one patient had virus with mutations associated with resistance to the self-reported treatment regimen at the time of treatment failure. This finding suggests that virologic failure associated with incarceration may be due to the development of drug resistance mutations in about half of cases. This implies that actions to prevent drug resistance, such as preventing treatment interruption during and after incarceration, may reduce virologic failure assoc.
