Fig. 3

Linker mediated HIV integration site assay (ISA) workflow. Total genomic DNA is first extracted then randomly sheared by Covaris sonification into 300–500 bp fragments. Sheared fragments are end repaired and a single dA overhang is added, then linkers containing a single T overhang are ligated onto the sheared ends (red). The pop out displays the PCR amplification strategy to selectively amplify integration sites. Primers that are complementary to the 5′ HIV LTR in U3 (dark grey arrow) and the 3′ HIV LTR in U5 (light grey arrow) are combined with linker specific primers (red arrows). The resulting amplicons contain linker sequence, the random breakpoint (BP), and the HIV/host junction sequence at the integration site (IS). The amplicons are then subjected to Illumina Miseq paired end sequencing. Sequences obtained are run through a stringent bioinformatics pipeline to map the location of the integrated provirus against a reference host genome and to determine the distance to breakpoint. Identical integration sites from amplicons with different break points in the host genome are the result of clonally expanded cells, whereas identical integration sites from amplicons with identical break point distances arose during PCR amplification