An analytical pipeline for identifying and mapping the integration sites of HIV and other retroviruses

Abstract

Background: All retroviruses, including human immunodeficiency virus (HIV), must integrate a DNA copy of their genomes into the genome of the infected host cell to replicate. Although integrated retroviral DNA, known as a provirus, can be found at many sites in the host genome, integration is not random. The adaption of linker-mediated PCR (LM-PCR) protocols for high-throughput integration site mapping, using randomly-sheared genomic DNA and Illumina paired-end sequencing, has dramatically increased the number of mapped integration sites. Analysis of samples from human donors has shown that there is clonal expansion of HIV infected cells and that clonal expansion makes an important contribution to HIV persistence. However, analysis of HIV integration sites in samples taken from patients requires extensive PCR amplification and high-throughput sequencing, which makes the methodology prone to certain specific artifacts. Results: To address the problems with artifacts, we use a comprehensive approach involving experimental procedures linked to a bioinformatics analysis pipeline. Using this combined approach, we are able to reduce the number of PCR/ sequencing artifacts that arise and identify the ones that remain. Our streamlined workflow combines random cleavage of the DNA in the samples, end repair, and linker ligation in a single step. We provide guidance on primer and linker design that reduces some of the common artifacts. We also discuss how to identify and remove some of the common artifacts, including the products of PCR mispriming and PCR recombination, that have appeared in some published studies. Our improved bioinformatics pipeline rapidly parses the sequencing data and identifies bona fide integration sites in clonally expanded cells, producing an Excel-formatted report that can be used for additional data processing. Conclusions:We provide a detailed protocol that reduces the prevalence of artifacts that arise in the analysis of retroviral integration site data generated from in vivo samples and a bioinformatics pipeline that is able to remove the artifacts that remain.

HIV proviral DNA integration can drive T cell growth ex vivo

“…The frequency and distribution of proviral DNA integration into genomic DNA was assessed by the linker-mediated PCR integration site assay ( Fig. 1 D ), incorporating a shearing step ( 2 , 4 ) to assess the relative frequency of descendants of single infected cells. In all, 202,179 and 524,691 total integration sites were mapped from donors 1 and 2, respectively ( Fig.…”

“…DNA was sheared by sonication, and subjected to linker-mediated PCR and paired-end sequencing using an Illumina HiSeq platform. Results were analyzed according to established ISA workflows ( 2 , 4 ).…”

Integration in or Near Oncogenes Plays Only a Minor Role in Determining the in Vivo Distribution of HIV Integration Sites Before or During Suppressive Antiretroviral Therapy

“…Total DNA was extracted from the PBMC two days following infection, which should not allow time for selection for, or against, proviruses integrated in specific sites or specific genes [29]. We used linker-mediated PCR [30] as previously described [21, 31] to identify IS. When we compared the global distribution of the IS from the PBMC obtained from the two individuals, the correlation coefficient was 0.98 and, for the rest of the analyses, we combined the data from the two sets of IS.…”

“…By combining in vivo IS from several studies [18, 21, 32, 33] (Table S1), we were able to assemble datasets comprising a total of 15,780 IS from pre-ART samples from 30 HIV-infected donors (hereafter referred to as “donors”) and 53,945 IS from on-ART samples from 39 donors (Table 1, S1). The introduction of a shearing step prior to linker ligation and PCR [30] creates different break points in flanking host DNA, making it possible to distinguish identical IS that arose by clonal expansion of a single infected cell [21, 30, 31]. After collapsing the IS with multiple breakpoints to a unique site, the two libraries contained 13,324 unique IS pre-ART and 33,336 unique IS on-ART (Table 1).…”

Correction to: An analytical pipeline for identifying and mapping the integration sites of HIV and other retroviruses

“…Following publication of the original article [ 1 ], it was reported that Additional file 1 was missing the supplemental figures (Figs. S1-S6) referenced in the file.…”