Investigation of Human Cancers for Retrovirus by Low-Stringency Target Enrichment and High-Throughput Sequencing

Abstract

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.

Mouse mammary tumor-like virus (MMTV) is present in human breast tissue before development of virally associated breast cancer

“…There are several possible reasons: (i) MMTV sequences identified by PCR can be inconsistent and false negatives are possible; (ii) MMTV sequences may be present in some, but not all parts of the tumor; (iii) although unlikely, changes in MMTV env sequences may have occurred. The problem of inconsistent outcomes of PCR based analyses has been considered in detail by Vinner et al [29]. There are particular difficulties in obtaining consistent outcomes from PCR analyses of retroviruses when present in extremely low viral concentrations.…”

“…These data, based on techniques very different from PCR, confirm the identification of MMTV-like gene sequences in human breast tumors and add validity to PCR based studies that were used in this current investigation. NGS techniques are not as sensitive for the identification of retroviral nucleotide sequences as PCR [29]. This is the reason for the much more frequent identification of MMTV-like nucleotide sequences by PCR compared to NGS.…”

Substitutions of short heterologous DNA segments of intragenomic or extragenomic origins produce clustered genomic polymorphisms

“…Research Ethics (Case 1304226). Both review boards approved the human research and waivered the requirement for informed consent, in accordance with national legislation (Sundhedsloven) (36).…”

“…For humans, we isolated DNA from blood samples and colon cancer tissues from three individuals (36) and sequenced the DNA on an Illumina HiSeq 2000. We called clustered polymorphisms with donor molecules for SPDIR largely as described above (see Supporting Information for details).…”

Enteric Virome and Carcinogenesis in the Gut

“…Perhaps the most comprehensive study characterizing viruses from a variety of sample types associated with the development of various cancers utilized a diverse selection enrichment method targeting all major viral groups followed by high-throughput sequencing [96]. This study investigated total DNA and RNA, mRNA, retroviral [97] and vertebrate viral capture [98], as well as enrichment of small circular DNA capture [99].…”