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We excluded sequences with translational problems (i.e., frameshift or stop codons). Shannon Entropy analyses (http://www.hiv.lanl.gov/content/index) were done on sequence set 1. Sequence changes in geographic regions over time were examined using set 2. HIV adaptation in specific countries was estimated using set 3. Differences in amino acid frequencies in HIV Gag from patients with or without specific HLA alleles were estimated using set 4. We counted and calculated the amino acid frequencies at each HIV-1 Gag p24 position; each query HIV-1 gag sequence was pairwise aligned to the HXB2 reference strain and mutations were defined as changes relative to this sequence. Oxalosuccinic acid The frequency of mutations in the p24 gene was then calculated across each of the data sets 2�C4. Because we analyzed all HIV sequences in the database, our intraepitope mutation frequencies ( Table S4) might differ from those reported by smaller cohorts. The HIV-1 consensus sequences were obtained from the HIV database. HLA allelic frequencies were obtained from the Allele Frequency Database (Gonzalez-Galarza et?al., 2011) and (Assane et?al., 2010?and?Novitsky et?al., 2001). When more than one data set was available from a country, we used Ponatinib in vitro weighted mean values unless otherwise specified. For the analyses in Figure?1, we used HLA frequencies from the UK (UK1?= Leeds [n?= 5,024], UK2?= Sheffield [n?= 4,755]), the US (US1?= Los Angeles, Seattle, and Detroit [n?= 1,070], US2?= Bethesda [n?= 307]), Germany (n?= 11,407), France (n?= 130), South Africa (Zulu, n?= 100; Xhosa, n?= 50), Zambia (n?= 256), Zimbabwe (n?= 108), Botswana (n?= 161), and Mozambique (n?= 250). We labeled peptide fragments as CD8 epitopes based on the HIV immunology database (http://www.hiv.lanl.gov/content/immunology). MI-773 research buy Because studies disagree as to whether HLA-Cw?03 can bind the VL10 epitope (Zappacosta et?al., 1997?and?Zarling and Lee, 1998), we did not include a selection pressure from HLA-Cw?03 in our analyses. We used multilevel modeling to examine the effects of HLA frequency, HIV region, and ethnic group on epitope yield. Further analyses were done using binomial generalized linear models (McCullough and Nelder, 1989) using the canonical logit link function, Fisher��s exact test, t tests, and a nonparametric sign test as described in the text and in the Supplemental Experimental Procedures. These analyses were done using GraphPad Prism 5 (GraphPad), the R?statistical package (version 3.0.0 [2013-04-03]), Microsoft Excel 2007 (Microsoft), and Stat Trek (Stattrek.com) software. A value of p?