Investigation of Human Cancers for Retrovirus by Low-Stringency Target Enrichment and High-Throughput Sequencing.
Although nearly one fifth of all human cancers have an infectious aetiology, the causes for the majority of cancers remain unexplained. Despite the enormous data output from high-throughput shotgun sequencing, viral DNA in a clinical sample typically constitutes a proportion of host DNA that is too small to be detected. Sequence variation among virus genomes complicates application of sequence-specific, and highly sensitive, PCR methods. Therefore, we aimed to develop and characterize a method that permits sensitive detection of sequences despite considerable variation. We demonstrate that our low-stringency in-solution hybridization method enables detection of <100 viral copies. Furthermore, distantly related proviral sequences may be enriched by orders of magnitude, enabling discovery of hitherto unknown viral sequences by high-throughput sequencing. The sensitivity was sufficient to detect retroviral sequences in clinical samples. We used this method to conduct an investigation for novel retrovirus in samples from three cancer types. In accordance with recent studies our investigation revealed no retroviral infections in human B-cell lymphoma cells, cutaneous T-cell lymphoma or colorectal cancer biopsies. Nonetheless, our generally applicable method makes sensitive detection possible and permits sequencing of distantly related sequences from complex material.
Centre for GeoGenetics Natural History Museum, University of Copenhagen Østervoldgade 5-7, 1350 Copenhagen K, Denmark.
Lasse
L
Vinner
Centre for GeoGenetics Natural History Museum, University of Copenhagen Østervoldgade 5-7, 1350 Copenhagen K, Denmark.
Tobias
T
Mourier
Center for Biological Sequence Analysis. Department of Systems Biology, Technical University of Denmark, Building 208, 2800 Lyngby, Denmark.
Jens
J
Friis-Nielsen
Department of Dermatology D92, Bispebjerg Hospital, Bispebjerg Bakke 23, 2400 N, Copenhagen, Denmark.
Robert
R
Gniadecki
Department of Heamatology Aalborg University Hospital, Sdr. Skovvej 15, 9000 Aalborg, Denmark.
Karen
K
Dybkaer
Department of Surgery Herlev Hospital, Herlev Ringvej 75, 2730, Herlev, Denmark.
Jacob
J
Rosenberg
Department of Pathology Herlev hospital, Herlev Ringvej 75, 2730, Herlev, Denmark.
Jill Levin
JL
Langhoff
Center for Biological Sequence Analysis. Department of Systems Biology, Technical University of Denmark, Building 208, 2800 Lyngby, Denmark.
David Flores Santa
DF
Cruz
Department of Microbiological Diagnostics and Virology, Statens Serum Institut Artillerivej 5, 2300 Copenhagen S, Denmark.
Jannik
J
Fonager
Center for Biological Sequence Analysis. Department of Systems Biology, Technical University of Denmark, Building 208, 2800 Lyngby, Denmark.
Jose M G
JM
Izarzugaza
Center for Biological Sequence Analysis. Department of Systems Biology, Technical University of Denmark, Building 208, 2800 Lyngby, Denmark.
Ramneek
R
Gupta
Center for Biological Sequence Analysis. Department of Systems Biology, Technical University of Denmark, Building 208, 2800 Lyngby, Denmark.
Thomas
T
Sicheritz-Ponten
Center for Biological Sequence Analysis. Department of Systems Biology, Technical University of Denmark, Building 208, 2800 Lyngby, Denmark.
Søren
S
Brunak
Centre for GeoGenetics Natural History Museum, University of Copenhagen Østervoldgade 5-7, 1350 Copenhagen K, Denmark.
Eske
E
Willerslev
1] Department of Epidemiology Research, Statens Serum Institut Artillerivej 5, 2300 Copenhagen S, Denmark [2] Health Sciences and Technology Aalborg University Fredrik Bajers Vej 7D2, 9220 Aalborg, Denmark.
Lars Peter
LP
Nielsen
Centre for GeoGenetics Natural History Museum, University of Copenhagen Østervoldgade 5-7, 1350 Copenhagen K, Denmark.
Anders Johannes
AJ
Hansen
D000818:Animals; D001483:Base Sequence; D015342:DNA Probes / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics; D015723:Gene Library; D015894:Genome, Human; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009369:Neoplasms / Q000821:virology*; D011533:Proviruses / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics*; D034421:Sus scrofa
D015342:DNA Probes; D004279:DNA, Viral; D012333:RNA, Messenger