title: Hybrid capture and next-generation sequencing identify viral integration sites from formalin-fixed, paraffin-embedded tissue.
abstract: Although next-generation sequencing (NGS) has been the domain of large genome centers, it is quickly becoming more accessible to general pathology laboratories. In addition to finding single-base changes, NGS allows for the detection of larger structural variants, including insertions/deletions, translocations, and viral insertions. We describe the use of targeted NGS on DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue, and show that the short read lengths of NGS are ideally suited to fragmented DNA obtained from FFPE tissue. Further, we describe a novel method for performing hybrid-capture target enrichment using PCR-generated capture probes. As a model, we captured the 5.3-kb Merkel cell polyomavirus (MCPyV) genome in FFPE cases of Merkel cell carcinoma using inexpensive, PCR-derived capture probes, and achieved up to 37,000-fold coverage of the MCPyV genome without prior virus-specific PCR amplification. This depth of coverage made it possible to reproducibly detect viral genome deletions and insertion sites anywhere within the human genome. Out of four cases sequenced, we identified the 5' insertion sites in four of four cases and the 3' sites in three of four cases. These findings demonstrate the potential for an inexpensive gene targeting and NGS method that can be easily adapted for use with FFPE tissue to identify large structural rearrangements, opening up the possibility for further discovery from archival tissue.
authors.affiliation: Department of Pathology, University of Utah, 500 Chipeta Way, Salt Lake, UT 84108, USA. eduncavage@me.com
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Duncavage
authors.forename: Vincent
authors.initials: V
authors.lastname: Magrini
authors.forename: Nils
authors.initials: N
authors.lastname: Becker
authors.forename: Jon R
authors.initials: JR
authors.lastname: Armstrong
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Demeter
authors.forename: Todd
authors.initials: T
authors.lastname: Wylie
authors.forename: Haley J
authors.initials: HJ
authors.lastname: Abel
authors.forename: John D
authors.initials: JD
authors.lastname: Pfeifer
mesh_terms: D001483:Base Sequence; D015266:Carcinoma, Merkel Cell / Q000235:genetics / Q000821:virology; D023061:Gene Order; D016679:Genome, Viral / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016612:Paraffin Embedding; D011120:Polyomavirus / Q000235:genetics; D027601:Polyomavirus Infections / Q000821:virology; D016707:Tissue Fixation; D014412:Tumor Virus Infections / Q000821:virology; D016662:Virus Integration / Q000235:genetics*