PCIP-seq

PCIP-seq maps viral integration sites and sequences host–virus junctions using pooled CRISPR cleavage, inverse long-range PCR, and Oxford Nanopore long-read sequencing.

Key Features:

• Circularization of DNA fragments: Host genomic DNA fragments containing proviral DNA are circularized to enable inverse PCR and targeted CRISPR cleavage.
• Pooled CRISPR guide RNAs and Cas9 cleavage: A pool of guide RNAs directs Cas9 to selectively cleave circularized fragments that contain proviral sequences to enrich integration-site–containing molecules.
• Inverse long-range PCR: Inverse long-range PCR amplifies regions flanking viral integration sites to capture both host and viral sequences at junctions.
• Long-read sequencing (Oxford Nanopore MinION): Amplified products are sequenced on the Oxford Nanopore MinION to generate long reads spanning viral genomes and integration junctions.

Scientific Applications:

• Virology and cancer research: Characterizes viral integration patterns relevant to disease progression and persistence of infected clones.
• Exogenous retroviruses: Applied to Human T-cell leukemia virus type 1 (HTLV-1), Bovine Leukemia Virus (BLV), and Human Immunodeficiency Virus type 1 (HIV-1) to study integration-driven clonal expansion and persistence.
• Endogenous retroviruses: Maps genomic locations and sequence variation of endogenous retroviral elements to study impacts on host genome evolution and function.
• Human papillomavirus (HPV): Maps HPV integration sites to investigate roles in carcinogenesis.

Methodology:

Sequencing and analysis of amplified products using Oxford Nanopore MinION long reads.