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Additional file 8: Figure S7. Size of the inducible peripheral HIV-1 reservoir in male and female HIV-infected individuals from Uganda (n = 62) and the U.S. (n =50) estimated by measuring cell-associated spliced HIV-1 RNA (EDITS assay) and proviral DNA. Unpaired t test was used to compare the reservoir size (number of cell equivalents per one million cells) between the different groups of patients. *p < 0.05, **p

Additional file 3: Figure S2. Sensitivity of EDITS primers. A Nested PCR primers nF6026 and nR6773 were evaluated using a serial dilution of DNA from the HIV-1 molecular clone pNL4-3 (0% to 100%) in a background of DNA from the non-HIV plasmid pUC19, at a final concentration of 0.1 ng/ml. DNA mixtures were amplified using Standard and Real-time PCR. Amplicons from the standard PCR were also deep sequenced and vpu/env mapped reads quantified using the DEEPGEN��� Software Tool Suite. Mean mapped reads and standard deviation are depicted. B ACH-2, a cell line latently infected with a copy of HIV-1 per cell, and MT-4, an HIV-negative human T cell line, were quantified and serial dilutions used to prepare seven mixtures containing 0 to 1000 CH-2 cells in a background of one million MT-4cells. Cell mixtures were ctivated with 100 ��g/ml of Concanavalin A and cell-associated spliced HIV-1 RNA quantified using EDITS as described in ���Methods��� section. Mean mapped reads and standard deviation are depicted.

Additional file 4: Figure S3. Analytical sensitivity of the EDITS assay. Jurkat E4, a cell line latently infected with a single copy of HIV-1 per cell, and Jurkat E6-1, an HIV-negative human T cell lymphoblast, were quantified and serial dilutions were used to prepare ten mixtures containing 0 to 400 Jurkat E4 cells in a background of one million Jurkat E6-1 cells in eight biological replicates. Cell mixtures were activated and cell-associated spliced HIV-1 RNA and proviral DNA quantified using EDITS assay as described in ���Methods��� section. Heatmaps indicate the number of HIV-1 vpu/end reads in each of the eight cell mixture replicates. Linear dynamic ranges and regression values (Pearson���s coefficient correlation) describing the relationship between mapped vpu/env reads and number of HIV positive cells in the mixtures are indicated. Median mapped reads and interquartile range are depicted.r, correlation coeficient; p, two-tailed p value.

Additional file 7: Figure S6. Quantification of number of cells used in the EDITS assay. Cellular DNA was extracted from one million memory CD4+ T cells purified from 15 Ugandan and 25 U.S. patients, as well as one million Jurkat E4 (HIV+)/Jurkat E^-1 (HIV-) cells from five points of the standard curve. One microlliter of cellular DNA was use to PCR amplify a 197 bp fragment of the cellular albumin gene. Amplicons were purified and quantified using Qubit 2.0 (Thermo Fisher Scientific). Unpaired t test was used to compare the concentration of albumin amplicon (ng/ul) between both cohort of patients and the cell mixture from the standard curve. Median values and interquartile range are depicted.

Additional file 6: Figure S5. Relevant metrics generated during each one of the deep sequencing runs corresponding to the EDITS assay and/or the proviral DNA test. Number of samples included in each specific MiSeq run. Clusters Passing Filter (Cluster PF) and Total Reads Passing Filters (Total Reads PF), represent the percentage of generated clusters and number of reads, respectively, that passed an internal quality filtering procedure used by Illumina. Cluster Density indicates the amount of clusters that were generated per flow cell surface area during the cluster generation stage. Error rate was calculated based on the PhiX control samples spiked in each deep sequencing run, showing the number of bases with a mismatch relative to the PhiX sequence. Median values and interquartile range are depicted.

Additional file 2: Figure S1. Specificity of EDITS primers. A Thirty-two HIV-1 group M isolates, as well as eight RNA or DNA viruses (BKV, BK virus; CMV, Cytomegalovirus; HSV-1 and HSV-2, Herpes simplex virus 1 and 2; VZV, Varicella zoster virus; HBV, Hepatitis B virus; HCV, Hepatitis C virus; and EBV, Epstein-Barr virus) were used to RT-PCR amplify a vpu/env 369 bp fragment and used to construct a neighbor-joining phylogenetic tree as described in ���Methods��� section. HIV-1 subtype-specific clusters are depicted. Bootstrap resampling (1000 data sets) of the multiple alignments, with percentage values above 75% are indicated by an asterisk. s/nt, substitutions per nucleotide. B The same 369 bp amplicons were deep sequenced, analyzed and reads quantified using the DEEPGEN��� Software Tool Suite.

Additional file 5: Figure S4. Validation of the EDITS assay. Memory CD4+ T cells from five ART-experienced, well-suppressed (plasma HIV-1 RNA load < 20 copies/ml) individuals infected with HIV-1 subtype B strains in Cleveland, OH were used to quantify the peripheral HIV-1 reservoir using the EDITS assay, proviral DNA deep sequencing assay, and a dPCR test designed to quantify proviral DNA as described in ���Methods��� section. Linear dynamic ranges and regression values (Pearson���s coefficient correlation) describing the relationship between the size of the HIV-1 reservoir quantified by each assay are indicated. r, correlation coeficient; p, two-tailed p value.