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

Maria Artesi, Vincent Hahaut, Fereshteh Ashrafi, Ambroise Marçais, Olivier Hermine, Philip Griebel, Natasa Arsic, Frank van der Meer, Arsène Burny, Dominique Bron, Carole Charlier, Michel Georges, Anne Van den Broeke, Keith Durkin
doi: https://doi.org/10.1101/558130
Maria Artesi
1Unit of Animal Genomics, GIGA-R, Université de Liège (ULiège), Avenue de l’Hôpital 11, B34, Liège 4000, Belgium.
2Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Boulevard de Waterloo 121, Brussels 1000, Belgium.
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Vincent Hahaut
1Unit of Animal Genomics, GIGA-R, Université de Liège (ULiège), Avenue de l’Hôpital 11, B34, Liège 4000, Belgium.
2Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Boulevard de Waterloo 121, Brussels 1000, Belgium.
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Fereshteh Ashrafi
1Unit of Animal Genomics, GIGA-R, Université de Liège (ULiège), Avenue de l’Hôpital 11, B34, Liège 4000, Belgium.
3Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
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Ambroise Marçais
4Service d’hématologie, Hôpital Universitaire Necker, Université René Descartes, Assistance Publique Hôpitaux de Paris, Paris, France.
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Olivier Hermine
4Service d’hématologie, Hôpital Universitaire Necker, Université René Descartes, Assistance Publique Hôpitaux de Paris, Paris, France.
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Philip Griebel
5Vaccine and Infectious Disease Organization, VIDO-Intervac, University of Saskatchewan, 120 Veterinary Road, Saskatoon, Canada S7N 5E3
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Natasa Arsic
5Vaccine and Infectious Disease Organization, VIDO-Intervac, University of Saskatchewan, 120 Veterinary Road, Saskatoon, Canada S7N 5E3
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Frank van der Meer
6Faculty of Veterinary Medicine: Ecosystem and Public Health, Calgary, AB, Canada
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Arsène Burny
2Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Boulevard de Waterloo 121, Brussels 1000, Belgium.
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Dominique Bron
2Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Boulevard de Waterloo 121, Brussels 1000, Belgium.
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Carole Charlier
1Unit of Animal Genomics, GIGA-R, Université de Liège (ULiège), Avenue de l’Hôpital 11, B34, Liège 4000, Belgium.
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Michel Georges
1Unit of Animal Genomics, GIGA-R, Université de Liège (ULiège), Avenue de l’Hôpital 11, B34, Liège 4000, Belgium.
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Anne Van den Broeke
1Unit of Animal Genomics, GIGA-R, Université de Liège (ULiège), Avenue de l’Hôpital 11, B34, Liège 4000, Belgium.
2Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Boulevard de Waterloo 121, Brussels 1000, Belgium.
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  • For correspondence: anne.vandenbroeke@bordet.be kdurkin@uliege.be
Keith Durkin
1Unit of Animal Genomics, GIGA-R, Université de Liège (ULiège), Avenue de l’Hôpital 11, B34, Liège 4000, Belgium.
2Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Boulevard de Waterloo 121, Brussels 1000, Belgium.
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  • For correspondence: anne.vandenbroeke@bordet.be kdurkin@uliege.be
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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.

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Posted February 22, 2019.
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Pooled CRISPR Inverse PCR sequencing (PCIP-seq): simultaneous sequencing of retroviral insertion points and the associated provirus in thousands of cells with long reads
Maria Artesi, Vincent Hahaut, Fereshteh Ashrafi, Ambroise Marçais, Olivier Hermine, Philip Griebel, Natasa Arsic, Frank van der Meer, Arsène Burny, Dominique Bron, Carole Charlier, Michel Georges, Anne Van den Broeke, Keith Durkin
bioRxiv 558130; doi: https://doi.org/10.1101/558130
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Pooled CRISPR Inverse PCR sequencing (PCIP-seq): simultaneous sequencing of retroviral insertion points and the associated provirus in thousands of cells with long reads
Maria Artesi, Vincent Hahaut, Fereshteh Ashrafi, Ambroise Marçais, Olivier Hermine, Philip Griebel, Natasa Arsic, Frank van der Meer, Arsène Burny, Dominique Bron, Carole Charlier, Michel Georges, Anne Van den Broeke, Keith Durkin
bioRxiv 558130; doi: https://doi.org/10.1101/558130

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