Identification of drug- resistance mutations in majority and minority subpopulations of HIV-1 quasispecies

The response of human immunodeficiency virus type 1 (HIV-1) quasispecies to antiretroviral therapy is influenced by the ensemble of mutants that composes the evolving population. Low-abundance subpopulations within HIV-1 quasispecies may determine the viral response to the administered drug combinations. However, routine sequencing assays available to clinical laboratories do not recognize HIV-1 minority variants representing less than 25% of the population. Although several alternative and more sensitive genotyping techniques have been developed, including next-generation sequencing (NGS) methods, they are usually very time consuming, expensive and require highly trained personnel, thus becoming unrealistic approaches in daily clinical practice. Here we describe the development and testing of a HIV-1 genotyping DNA microarray that detects and quantifies, in majority and minority viral subpopulations, relevant mutations and amino acid insertions in 42 codons of the pol gene associated with resistance and multidrug resistance to protease (PR) and reverse transcriptase (RT) inhibitors. A customized bioinformatics protocol has been implemented to analyze the microarray hybridization data by including a new normalization procedure and a stepwise filtering algorithm which resulted in the highly accurate (96.33%) detection of positive/negative signals. This microarray has been tested with 57 subtype B HIV-1 clinical samples extracted from multi-treated patients, showing an overall identification of 95.53% and 89.24% of the queried PR and RT codons, respectively, and enough sensitivity to detect minority subpopulations representing as low as 5-10% of the total quasispecies. Such a genotyping platform represents an efficient diagnostic and prognostic tool useful to personalize antiviral treatments in clinical practice. We describe a novel genotyping DNA microarray platform that contains a panel of 160 optimized oligonucleotide probes complementary to HIV PR and RT genomic regions involved in resistance to NRTIs, NNRTIs and PIs. This platform has been developed using 17 clonal HIV-1 samples and selected mixtures of them at different ratios, and further tested with 57 HIV-1 clinical samples extracted from multi-treated infected patients. Please note that [1] The array has two blocks that can be independently hybridized and thus, several raw data files were duplicated with the file name correponding to each sample (indicated in the sample description field). For example, 6.95c8-PRRT-2.1 normalized data is derived from Block/Array Column: 1 (in 6.95c8-PRRT-2.1.csv) and 6.95c8-PRRT-2.2 from Block/Array Column: 2 (in the same raw file but renamed as 6.95c8-PRRT-2.2.csv). In the normalized matrix, non corresponding block is filled with the 'null' label. [2] The Sequences reported in the sample 'Characteristics:Sequence(s)' column are the PR, RT or PR-RT regions from the considered wild type proteins or the mutated counterparts, cloned into plasmids, used to characterize the hybridization signal of the probes, or the consensus sequences extracted from the patients. [3] Some of the arrays have been analyzed with the ScanArray software, and the rest with the GenePix software (as indicated in the 'characteristics: Scanner' column). [4] Probes positions in GenePix raw files are identified by labels 'Block','Row' and 'Column', while the equivalent labels in ScanArray raw files are 'Array Column','Spot Row' and 'Spot Column'. [5] The number of rows in each raw data file depends on 3 factors, the scanner used (GenePix or ScanArray), the included regions (PR,RT or PRRT) and if it includes 1 or 2 blocks. For example, pWT_pINS-RT-0-1.csv = GenePix file including PR-RT regions and 2 blocks (800 rows) pWT-RT-7.2.csv = ScanArray file with only the RT region and 1 block (325 rows) A47-PR-1.csv = ScanArray file with only the PR region and 1 block (157 rows) 5.96c9-PRRT-1.1.csv = ScanArray file with the PR-RT regions and 1 block (421 rows) 5.96c9-PRRT-1.2.csv = ScanArray file with the PR-RT regions and 2 blocks (781 rows)