Pooled CRISPR Inverse PCR sequencing (PCIP-seq): simultaneous sequencing of retroviral insertion points and the associated provirus in thousands of cells with long reads

Abstract

Retroviral infections create a large population of cells, each defined by a unique proviral insertion site. Methods based on short-read high throughput sequencing can identify thousands of insertion sites, but the proviruses within remain unobserved. We have developed Pooled CRISPR Inverse PCR sequencing (PCIP-seq), a method that leverages long reads on the Oxford Nanopore MinION platform to sequence the insertion site and its associated provirus. We have applied the technique to three exogenous retroviruses, HTLV-1, HIV-1 and BLV, as well as endogenous retroviruses in both cattle and sheep. The long reads of PCIP-seq improved the accuracy of insertion site identification in repetitive regions of the genome. The high efficiency of the method facilitated the identification of tens of thousands of insertion sites in a single sample. We observed thousands of SNPs and dozens of structural variants within proviruses and uncovered evidence of viral hypermutation, recombination and recurrent selection.

An Improved Sequencing-Based Bioinformatics Pipeline to Track the Distribution and Clonal Architecture of Proviral Integration Sites

“…Surprisingly, NGS mapping of proviral integration sites of ATL 2 revealed two major HTLV-1 insertions in chr5 and chr16 with a relative abundance of 49.5 and 36.3%, respectively, and a third minor integration site in chr1 representing 13.6% ( Figure 5A). In contrast, the application of PCIP-seq, an Oxford Nanopore-based long-read sequencing method developed by us to capture proviral integration sites and simultaneously sequence the viral genome associated with each site (Artesi et al, 2019), revealed three major proviruses located on chr5, chr16, and chr1 in ATL 2, each responsible for 33.7, 34.5, and 31.8% of the HTLV-1/host hybrid reads, respectively. These data suggested the presence of three proviral integrations in a single T-cell clone.…”

Emerging PCR-Based Techniques to Study HIV-1 Reservoir Persistence

“…In their preprint, the researchers present a proof-of-concept of this protocol on two HIV-1-infected patients on ART. They showcased the recovery of several integration sites like STAT5B (also found via ISS-and MDA-based techniques) and the (partial) genomes of the associated proviruses, enabling them to distinguish intact from defective viruses and detect the occurrence of clonally expanded cells [12,13,[18][19][20]. Further optimization and increasing of sensitivity could lead to a novel approach while avoiding the use of limiting dilutions, which would make the protocol less laborious and expensive, thus offering a huge improvement on the second technical challenge.…”

“…Both technologies are maturing at a vast rate, with the associated error rate becoming less of an issue [36]. Exploration has been done in terms of applicability within the HIV-1 context, for example, by Artesi et al [20]. In this preprint, the authors present a technique called pooled CRISPR inverse PCR sequencing (PCIP-seq) to asses retroviral integration and proviral genomes, mirroring the outcome of MDA-based techniques [20].…”

“…Exploration has been done in terms of applicability within the HIV-1 context, for example, by Artesi et al [20]. In this preprint, the authors present a technique called pooled CRISPR inverse PCR sequencing (PCIP-seq) to asses retroviral integration and proviral genomes, mirroring the outcome of MDA-based techniques [20]. In short, genomic DNA is extracted, randomly sheared into fragments, and circularized, after which a CRISPR-guided enrichment strategy is employed to select for circles containing infected provirus.…”