Ried overnight at room temperature, individually packaged in a zip-lock bag and stored at 220uC with desiccant. After centrifugation, plasma was transferred to a fresh tube and the pellet re-suspended and used for peripheral blood mononuclear cells (PBMC) isolation using FicollHypaque density gradient centrifugation. Nucleic acid (NA) was extracted from two DBS spots and from 200 ml of plasma using the Nuclisens EasyMag system (Biomerieux, Canada) following manufacturer’s instructions. Cellular DNA was extracted from the isolated PBMCs using QIAamp DNA Mini Kit (Qiagen, Mississauga, Canada). Plasma VL was measured using the Versant HIV RNA 3.0 Assay (bDNA, Siemens Healthcare Diagnostics, Mississauga, Canada).HIV Genotyping by TPPTPP was performed on PCR amplified, multiplex identifier (MID) labeled amplicons, covering the protease (PR) and reverse transcriptase (RT) (partial) genes from NA extracts derived from DBS, plasma, or PBMC following order Doramapimod published methods [4].Sequence analysisTPP reads were screened for quality using the GS FLX defaults and decoded using Roche Amplicon Variant Analyzer software. Reads passing initial QC were re-screened using custom Perl scripts to further improve the accuracy of the downstream analysis [4,16,17] (Figure S1). In brief, the reads were the first filtered using the following criteria: 1) a read length of 100 bps; 2) an average quality score of 25; 3) no ambiguous bases present in the read. All valid reads were then mapped to the HXB2 DLS 10 reference (GenBank Accession: K03455) by BLAST. Only reads that had 65 overlap and 75 identity with HXB-2 reference were employed to generate multiple alignments on assumption that there are not true insertions. The net PCR and pyrosequencing error rates were estimated by parallel pyrosequencing three pedigreed plasmid controls. Sequence contigs for each specimen were built using all the valid reads which were aligned against HXB-2. Two consensus sequences were generated for each specimen with mixed base identification thresholds (MBIT) of 5 and 20 respectively. The MBIT defines the threshold for calling a minor variant based upon the frequency of the mutation at a specific locus within the aligned individual pyrosequencing reads. A 5 MBIT was chosen as the reference consensus sequence in order to maximize our ability to detect discordance among the different specimen formats. Discordant base positions from any of the three specimen formats from the same subject were flagged. These flagged positions were then used to evaluate the overall inter-format sequence concordance rates (SCR) among specimens by using the 20 MBIT to simulate the readout from conventional genotyping. The derived SCRs were analyzed using SPSS 12.0, stratified according VL,Decoding DBS Genotype of HIV with TPPTable 1. Demographic and clinical characteristics data of subjects in the study cohort.Subjects 013 020 022 024 029 036 038 039 042 049 058 064 065 067 080 083Viral Load (Copies/ml) 6,080 3,406 9,356 38,658 43,750 5,748 46,896 2,378 1,448 7,352 2,278 35,774 14,240 11,076 4,462 504 50,CD4 Count (per ml) 282 587 250 316 307 535 450 108 484 185 499 444 324 256 208 49Sexa M M M M M F M M F M M M M M M M MAge Group 40?0 18?5 40?0 .60 25?0 18?5 25?0 40?0 40?0 25?0 25?0 40?0 40?0 40?0 25?0 40?0 40?Birth Placeb N. A N. A S. A N. A Africa N. A N.A N.A N.A S.A N.A N.A N.A N.A S.A N.A N.AHIV-1 Subtype B B B B C B B B B B B B B B B B BDuration of Infection (yr) 2.0 5.5 8.0 6.5 4.5 1.0 5.0 20.0 18.0 0.Ried overnight at room temperature, individually packaged in a zip-lock bag and stored at 220uC with desiccant. After centrifugation, plasma was transferred to a fresh tube and the pellet re-suspended and used for peripheral blood mononuclear cells (PBMC) isolation using FicollHypaque density gradient centrifugation. Nucleic acid (NA) was extracted from two DBS spots and from 200 ml of plasma using the Nuclisens EasyMag system (Biomerieux, Canada) following manufacturer’s instructions. Cellular DNA was extracted from the isolated PBMCs using QIAamp DNA Mini Kit (Qiagen, Mississauga, Canada). Plasma VL was measured using the Versant HIV RNA 3.0 Assay (bDNA, Siemens Healthcare Diagnostics, Mississauga, Canada).HIV Genotyping by TPPTPP was performed on PCR amplified, multiplex identifier (MID) labeled amplicons, covering the protease (PR) and reverse transcriptase (RT) (partial) genes from NA extracts derived from DBS, plasma, or PBMC following published methods [4].Sequence analysisTPP reads were screened for quality using the GS FLX defaults and decoded using Roche Amplicon Variant Analyzer software. Reads passing initial QC were re-screened using custom Perl scripts to further improve the accuracy of the downstream analysis [4,16,17] (Figure S1). In brief, the reads were the first filtered using the following criteria: 1) a read length of 100 bps; 2) an average quality score of 25; 3) no ambiguous bases present in the read. All valid reads were then mapped to the HXB2 reference (GenBank Accession: K03455) by BLAST. Only reads that had 65 overlap and 75 identity with HXB-2 reference were employed to generate multiple alignments on assumption that there are not true insertions. The net PCR and pyrosequencing error rates were estimated by parallel pyrosequencing three pedigreed plasmid controls. Sequence contigs for each specimen were built using all the valid reads which were aligned against HXB-2. Two consensus sequences were generated for each specimen with mixed base identification thresholds (MBIT) of 5 and 20 respectively. The MBIT defines the threshold for calling a minor variant based upon the frequency of the mutation at a specific locus within the aligned individual pyrosequencing reads. A 5 MBIT was chosen as the reference consensus sequence in order to maximize our ability to detect discordance among the different specimen formats. Discordant base positions from any of the three specimen formats from the same subject were flagged. These flagged positions were then used to evaluate the overall inter-format sequence concordance rates (SCR) among specimens by using the 20 MBIT to simulate the readout from conventional genotyping. The derived SCRs were analyzed using SPSS 12.0, stratified according VL,Decoding DBS Genotype of HIV with TPPTable 1. Demographic and clinical characteristics data of subjects in the study cohort.Subjects 013 020 022 024 029 036 038 039 042 049 058 064 065 067 080 083Viral Load (Copies/ml) 6,080 3,406 9,356 38,658 43,750 5,748 46,896 2,378 1,448 7,352 2,278 35,774 14,240 11,076 4,462 504 50,CD4 Count (per ml) 282 587 250 316 307 535 450 108 484 185 499 444 324 256 208 49Sexa M M M M M F M M F M M M M M M M MAge Group 40?0 18?5 40?0 .60 25?0 18?5 25?0 40?0 40?0 25?0 25?0 40?0 40?0 40?0 25?0 40?0 40?Birth Placeb N. A N. A S. A N. A Africa N. A N.A N.A N.A S.A N.A N.A N.A N.A S.A N.A N.AHIV-1 Subtype B B B B C B B B B B B B B B B B BDuration of Infection (yr) 2.0 5.5 8.0 6.5 4.5 1.0 5.0 20.0 18.0 0.
