RT Journal Article
SR Electronic
T1 Pooled CRISPR Inverse PCR sequencing (PCIP-seq): simultaneous sequencing of retroviral insertion points and the associated provirus in thousands of cells with long reads
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 558130
DO 10.1101/558130
A1 Maria Artesi
A1 Vincent Hahaut
A1 Fereshteh Ashrafi
A1 Ambroise Marçais
A1 Olivier Hermine
A1 Philip Griebel
A1 Natasa Arsic
A1 Frank van der Meer
A1 Arsène Burny
A1 Dominique Bron
A1 Carole Charlier
A1 Michel Georges
A1 Anne Van den Broeke
A1 Keith Durkin
YR 2019
UL http://biorxiv.org/content/early/2019/02/22/558130.abstract
AB 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.