Abstract
BackgroundAll 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.ResultsTo 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.ConclusionsWe 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.
Highlights
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
The protocol we describe here has been optimized to amplify the relatively rare HIV integration sites present in samples taken from patients
We present both a selective amplification protocol designed to reduce the artifacts commonly arising during linker-mediated PCR protocols that involve high levels of amplification and a matching bioinformatics pipeline designed to remove the residual artifacts
Summary
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. Despite contradictory claims in the literature [21], it is clear that clonally expanded cells can carry an intact infectious provirus and can release low levels of infectious virus into the blood [22]. It appears that the majority of the proviruses in clones of expanded cells are transcriptionally silent [23] explaining how some infected cells can survive and expand into clones. This reactivation leads to a rapid rekindling of the infection if ART is interrupted [24, 25]
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