Ewer systemic basecalling sequencing errors, increased multiplexing, use of a FDA-approved
Ewer systemic basecalling sequencing errors, increased multiplexing, use of a FDA-approved sequencing platform, detection of drug resistance to integrase inhibitors in addition to reverse transcription inhibitors and protease inhibitors with a single amplicon, and finally, simultaneous genotyping of all major group M HIV subtypes with a single set of amplification primers. Detection of Integrase inhibitor drug resistance is particularly noteable because integrase inhibitors are now recommended by the department of Health and Human Services as part of first-line antiretroviral therapy in the United States [8]. The use of integrase inhibitors as part of initial antiretroviral therapy is expected to increase because of the recent FDA approval of a new once daily integrase inhibitor (elvitegravir) that can be co-formulated with emtricitabine, tenofovir disoproxyl fumarate and cobicistat as a single pill once daily regimen (STRIBILD) [9]. Current genotyping methods used to detect drug resistance to integrase inhibitors are limited and typically involve a separate assay from that used to identify drug resistance to reverse transcriptase and protease inhibitors [10,11]. However, due to proximity of the integrase Stattic site protein-coding region to the reverse transcriptase and protease protein-coding region in the viral genome, it is feasible to incorporate drug resistance genotyping of all three gene products together from a single PCR amplicon. By using a single amplicon, no additional labor or expense is added to the assay already detecting protease and reverse transcriptase drug resistance mutations. The FDA-approved HIV drug resistance genotyping assays currently in use were developed using HIV group M subtype B viruses [12]. However, subtype C comprises about half of the worldwide epidemic followed by subtype A (12 ) and then B (11 ) [13]. Current commercial genotypingassays perform rather poorly with non-subtype B viruses because seven sets of sequencing primers must bind to highly variable targets during sequencing [12,14]. Therefore, non-B assays require more replication and for many samples, fail altogether with the current commercially available genotyping assays. There have been many successful adaptations of the commercial Sanger-based genotyping assay to improve genotyping of non-B HIV subtypes, however, these assays do not include PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28381880 detection of integrase inhibitor mutations and are less sensitive than next-generation sequencing approaches [15-21]. Cost is a major barrier to drug resistance genotyping in resource-limited settings. Deep sequencing technologies have significantly reduced the cost of sequencing but are not yet incorporated into standard HIV drug resistance genotyping. An Illumina MiSeq sequencer can yield over 25 million sequencing reads per run with the latest technology (MiSeq reagent kit v3). Through multiplexing, 96 samples can be processed in one run, bringing the cost of sequencing alone to < 10/sample. This is 3-fold less than Sanger-based sequencing. In addition, the clonal nature of deep sequencing methods along with the large number of sequencing reads generated improves sensitivity for the detection of mutations compared to PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28724915 Sanger-based sequencing [12,22,23]. Lastly, the Illumina MiSeq platform is currently the least error-prone deep sequencing method [24,25].Results and discussionPCR primers amplify major group M HIV subtypes A, B, C, D, CRF01_AE and CRF02_AGHIV sequences from all subtypes found in the Los Alamos N.