1 Supplementary Methods: Total, integrated, and episomal 2-LTR circles were quantified by real-time quantitative PCR (qPCR), using the methods described below. Primers and probes used in the various qPCR are detailed in supplementary table 1. Calculated HIV DNA copy numbers were normalized for DNA input by measurement of beta-actin by qPCR, and expressed as HIV DNA copies per 106 cells. Approximately 500ng DNA was assayed per replicate for each qPCR. This equates to 80,000 cells, thus the limit of detection (LOD) for each HIV DNA assay, was determine to be 1 copy per 80,000 cells or, 12.5 copies per 106 cells. The limit of quantification (LOQ) for each HIV DNA qPCR was determined by multiplying the LOD by the lower range of the standard curve used to quantify test samples (total and 2-LTR 125 copies or integrated 100 copies per 106 cells). The standard curves for the individual HIV DNA qPCR are detailed below. Total HIV DNA qPCR Total HIV DNA was quantified by qPCR using a primer and probe set targeting the HIV gag gene. All samples were assayed in duplicate and total HIV DNA copies determined using a set of plasmid standards. All HIV DNA qPCR assays were performed on an iQTM5 real-time PCR Detection System (Bio-Rad; California, USA). Reactions contained; 12.5µl iQ Supermix, 800nM SK145 and SKCC1B, 200nM SKLNA2, 5µl DNA template and dH2O to a final volume of 25µl. Reactions were subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. For patient 11001 reactions contained: 12.5µl iQ Supermix, 800nM 11001 Forward and 11001 Reverse, 300nM 11001 TG Probe, 5µl DNA template, and dH2O to a final volume of 25µl. Reactions were subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 64.5°C for 1 minute. Standard curves were generated using the pNL4-3 plasmid, obtained through the AIDS Research and Reference Reagent Program (catalogue number 114), Division of AIDS, NIAID, NIH from Dr. Malcolm Martin [1]. The pNL4-3 plasmid was linearised by EcoRI (New England Biolabs; 2 Massachusetts, USA) restriction enzyme digestion, and linearisation confirmed by agarose gel electrophoresis. Plasmid DNA concentration was determined by nano-spectrophotometry, and plasmid copy number calculated based on the nucleotide length of the pNL4-3 plasmid, and the weight of the individual nucleotides. 10-fold serial dilutions were made 107 to 10 copies per 5µl and stored as 11µl aliquots at -80°C for use in individual qPCR assays. For patient 11001: a standard curve was designed by generating a plasmid containing patient specific PCR product spanning the region of the HIV gag gene targeted by the primer and probe set. 10-fold serial dilutions were made from 107 to 10 copies per 5µl, and dilutions stored as 11µl aliquots at -80°C for use in individual qPCR assays. For use a positive control, genomic DNA was extracted from PBMC isolated from an HIV+ individual, using the QIAGEN DNeasy Blood and Tissue Kit. Extracted DNA was dispensed in 21µl aliquots and stored at -80°C for use in individual qPCR assays. Integrated HIV DNA qPCR Integrated HIV DNA levels were quantified by a nested qPCR [2, 3]. This assay specifically amplifies integrated HIV DNA by utilizing the Alu repeat element of the human genome. The 1 st round primers target the HIV ltr (L-M667) and human genomic Alu repeat elements (Alu 1 and Alu 2), to specifically amplify integrated HIV DNA. The two Alu-specific primers allow for the amplification of integrated HIV DNA regardless of its orientation relative to the Alu repeat elements. The 2nd round primers target the heel of L-M667 (Lambda T) and the HIV ltr (AA55M) to specifically amplify 1st round products. The 2nd round qPCR includes a probe (LTR-FL) to quantify HIV DNA by qPCR. During the 1st round PCR, test samples were assayed in quadruplicate, and standards in a single replicate. 1st round products were then measured in duplicate by the 2nd round qPCR. A standard curve generated from the HIV infected T cell clone ACH-2 was used to calculate integrated HIV DNA copies in test samples. For patients 5004 and 11004, individual primers, probe, and plasmid standards were designed and optimized to compensate for differences in these patients HIV DNA 3 sequences. 1st round reactions were performed on a Veriti® Thermal Cycler (Life Technologies; California, USA) or T100TM Thermal Cycler (Bio-Rad; California, USA). 2nd round reactions were performed on an iQTM5 real-time PCR Detection System (Bio-Rad; California, USA). For the standard integrated HIV DNA qPCR, 1st round reactions contained: 12.5µl iQ Supermix, 300nM Alu 1 and Alu 2, 100nM L-M667, 1µM MgCl2, 5µl DNA template, and dH2O to a final volume of 25µl. 1st round reactions were subject to: 94°C for 7 minutes, 12 cycles of 94°C for 30 seconds, 60°C for 30 seconds then 72°C for 3 minutes, then 72°C for 7 minutes. 2nd round reactions contained: 12.5µl iQ Supermix, 300nM Lambda T and AA55M, 200nM LTR-FL, 1µM MgCl2, 5µl 1st round product, and dH2O to a final volume of 25µl. 2nd round reactions were subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. For patient 5004 1st round reactions contained: 12.5µl iQ Supermix, 300nM Alu 1 and Alu 2 primers, 100nM L-M667, 1µM MgCl2, 5µl DNA template, and dH2O to a final volume of 25µl. 1st round reactions were subject to: 94°C for 7 minutes, 12 cycles of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 3 minutes, then 72°C for 7 minutes. 2nd round reactions contained: 12.5µl iQ Supermix, 300nM Lambda T and AA55M 2, 200nM LTR-FL 2, 1µM MgCl2, 5µl 1st round product, and dH2O to a final volume of 25µl. 2nd round reactions were subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. For patient 11004 1st round reactions: contained 12.5µl iQ Supermix, 300nM Alu 1 and Alu 2 primers, 100nM L-M667 2, 1µM MgCl2, 5µl DNA template, and dH2O to a final volume of 25µl. 1st round reactions were subject to: 94°C for 7 minutes, 12 cycles of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 3 minutes, then 72°C for 7 minutes. 2nd round reactions contained: 12.5µl iQ Supermix, 300nM Lambda T and AA55M 3, 200nM LTR-FL 3, 1µM MgCl2, 5µl 1st round product, and dH2O to a final volume of 25µl. 2nd round reactions were subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. To generate a standard curve serial dilutions were made using DNA extracted from a latently HIV infected T cell line (ACH-2). ACH-2 cells were obtained through the AIDS Research and 4 Reference Reagent Program (catalogue number 349), Division of AIDS, NIAID, NIH from Dr. Thomas Folks [4, 5]. DNA was extracted from ACH-2 cells using the QIAGEN DNeasy Blood and Tissue kit. DNA quantity and quality were assessed by nano-spectrophotometry. ACH-2 cells contain 1 copy of integrated HIV DNA per cell, therefore, assuming that 50ng of DNA is equal to the DNA content of 8,000 cells and hence contains 8,000 copies of integrated HIV DNA, 4-fold serial dilutions were made from 50ng (8,000 copies) to 0.012ng (2 copies) per 5µl. Serial dilutions were dispensed in 6µl aliquots and stored at -80°C for use in individual qPCR. For patients 5004 and 11004 standard curves were designed by generating plasmids containing patient specific PCR products spanning the HIV 3’LTR region targeted by the primer and probe sets. 10-fold serial dilutions were made from 107 to 10 copies per 5µl, and stored as 11µl aliquots at -80°C for use in individual qPCR. The positive control generated for the total HIV DNA qPCR was also used as the positive control for integrated HIV DNA qPCR. Episomal 2-LTR HIV DNA qPCR Episomal 2-LTR HIV DNA circles were quantified using a set of primers and probe specifically targeting the junction between the 5’ltr and 3’ltr region, unique to 2-LTR HIV DNA circles [6]. For patients 1005, 11003 and 11005, individual primer and probe sets and standards were designed and optimized to compensate for differences in these patients HIV DNA sequences. All samples were assayed in duplicate and 2-LTR HIV DNA copies determined using a set of plasmid standards. Reactions were performed on an iQTM5 real-time PCR Detection System (Bio-Rad; California, USA). For the standard 2-LTR HIV DNA qPCR reactions containe: 12.5µl iQ Supermix, 280nM 2-LTR J F and 2-LTR J R, 200nM 2-LTR Probe 1, 5µl DNA template, and dH2O to a final volume of 25µl. Reactions were subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. 5 For patient 1005 reactions contained: 12.5µl iQ Supermix, 280nM 2-LTR J F3 and HIV reverse, 200nM 2-LTR Probe 2, 5µl DNA template, and dH2O to a final volume of 25µl. Reactions were subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 62.8°C for 1 minute. For patient 11003 reactions contained: 12.5µl iQ Supermix, 280nM 2-LTR J F and HIV reverse, 200nM 2-LTR Probe 1, 5µl DNA template, and dH2O to a final volume of 25µl. Reactions were subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 65.1°C for 1 minute. For patient 11005 reactions contained: 12.5µl iQ Supermix, 280nM 2-LTR J F and 2-LTR J R2, 200nM 2-LTR Probe 1, 5µl DNA template and dH2O to a final volume of 25µl. Reactions were subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 67.2°C for 1 minute. A standard curve was generated using a plasmid containing PCR product spanning the 2-LTR junction. HUT78 cells, obtained through the AIDS Research and Reference Reagent Program (catalogue number 89), Division of AIDS, NIAID, NIH from Dr. Robert Gallo [7], were infected using pNL4-3 virus. DNA was extracted from pNL4-3 infected HUT78 cells using the QIAGEN DNeasy Blood and Tissue Kit. A PCR product spanning the 2-LTR junction specific to 2-LTR HIV DNA circles was amplified using this DNA. For patients 1005, 11003 and 11005, specific standard curves were designed by generating patient specific 2-LTR junction PCR products. 10fold serial dilutions of the various plasmid standards were made from 10 7 to 10 copies per 5µl. Dilutions were dispensed in 11µl aliquots and stored at -80°C for use in individual qPCR. For use as a positive control: DNA was extracted from HIV (pNL4-3) infected HUT78 cells, using the QIAGEN DNeasy Blood and Tissue kit. DNA quantity and quality was assessed by nanospectrophotometry. DNA was dispensed in 21µl aliquots and stored at -80°C for use in individual qPCR. Beta-Actin qPCR Beta-actin levels in extracted DNA were quantified using the TaqMan β–actin detection kit (Life Technologies; California, USA). The primers (Forward CGG AAC CGC TCA TTG CC, and 6 Reverse ACC CAC ACT GTG CCC ATC TA) and probe (FAM/TAMRA labelled) in this kit target a 289bp conserved region of the β-actin exon 3 [8]. Reactions contained: 12.5µl of iQ Supermix, 180nM of Forward and Reverse primers, 120nM probe, 5µl DNA template, and dH2O to a final volume of 25µl. Reactions were performed using an iQTM5 real-time PCR Detection system (Bio-Rad; California, USA) and were subject to: 1 cycle of 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. All samples were assayed in duplicate and DNA concentration determined using a set of DNA standards. To generate a standard curve serial dilutions were made using DNA extracted from pooled HIV negative PBMC. PBMC were isolated from HIV negative pooled buffy coats, provided by the Australian Red Cross Blood Service, by Ficoll-Paque separation. DNA was extracted from the isolated PBMC using the QIAGEN DNeasy Blood and Tissue Kit, and DNA quality and quantity were assessed by nano-spectrophotometry. 10-fold serial dilutions were made from 100ng to 0.01ng per µl, dispensed in 11µl aliquots, and stored at -80°C for use in individual qPCR. A positive control of pooled human DNA at a concentration of 10ng/µl was provided with the TaqMan Beta-Actin Detection Reagents Kit. The provided DNA solution was dispensed in 11µl aliquots and stored at -80°C for use in individual qPCR assays. 7 Supplementary Table 1: Primers and probes used for qPCR quantification of HIV DNA species Primer Probe qPCR / Sequence (5’ → 3’) 5’ mod. 3’ mod. bp Binding site* 22 1359-1380 17 1402-1418 SK145 AGT GGG GGG ACA TCA AGC AGC C SKLNA2 AT[C] A[A]T [G]AG GAA [G]CT [G]C SKCC1B TAC TAG TAG TTC CTG CTA TGT CAC TTC C 28 1486-1513 11001 Forward GGT GGG GGG ACA TCA AGC AGC CAT GCA AAT 30 1359-1388 11001 TG Probe AT[T] A[A]T [G]AG GAG [G]CT [G]C 17 1402-1418 11001 Reverse TAC TAG TGG TTC CTG CTA TGT CAC TTC C 28 1486-1513 2-LTR J F GCT AAC TAG GGA ACC CAC TGC TTA AG 26 9582-9607 2-LTR J F3 GCT AGC TAG AGA ACC CAC TGC TTA AG 26 9582-9607 2LTR 2-LTR Probe 1 ACA [C]A[C] A[A]G [G][T]T 6-FAM BHQ-1 12 58-69 HIV DNA 2-LTR Probe 2 ACA [C]G[C] A[A]G [G][C]T 6-FAM BHQ-1 12 58-69 2-LTR J R TGG GTG GTG CCT CAA ACT AGT ACC AGT 27 128-154 HIV reverse TGG ATG GTG CTA CAA GCT AGT ACC AGT 27 128-154 Alu 1 TCC CAG CTA CTG GGG AGG CTG AGG 24 n/a Alu 2 GCC TCC CAA AGT GCT GGG ATT ACA G 25 n/a L-M667 ATG CCA CGT AAG CGA AAC TCT GGC TAA CTA GGG AAC CCA CTG 42 9579-9607 L-M667 2 ATG CCA CGT AAG CGA AAC TCT GGC TAA CTA GTG AAC CCA CTG 42 9579-9607 Integrated AA55M GCT AGA GAT TTT CCA CAC TGA CTA A 25 9694-9718 HIV DNA AA55M 2 GCT AGA GAT TTT TCT ACT TCG ATT A 25 9694-9718 AA55M 3 GCT AGA GAT TTT CCA CAC TGA CTA TC 26 9694-9719 LTR-FL CAC AAC AGA CGG GCA CAC ACT ACT TGA 6-FAM TAMRA 27 9634-9660 LTR-FL 2 AAC AAC AGA CGG GCA CAC ACT ACT TGA 6-FAM TAMRA 27 9634-9660 LTR-FL 3 CAC AAC AGA TGG GCA CAC ACT ACT GA 6-FAM TAMRA 26 9634-9659 Lambda T1 ATG CCA CGT AAG CGA AAC T 19 n/a Total DNA HIV 6-FAM 6-FAM BHQ-1 BHQ-1 Red letters represent mismatched nucleotides from patient specific qPCR; Brackets indicate locked nucleic acids; mod = modification; 6-FAM = 6-Carboxyfluorescein; BHQ-1 = Black Hole Quencher; TAMRA = Tetramethylrhodamine; bp = base pairs. *Relative to HIV B reference genome (HXB2). 1Binds to the underlined heel of L-M667/LM667 #2. 8 1. 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