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Virus expression detection reveals RNA-sequencing contamination in TCGA.

ABSTRACT: BACKGROUND:Contamination of reagents and cross contamination across samples is a long-recognized issue in molecular biology laboratories. While often innocuous, contamination can lead to inaccurate results. Cantalupo et al., for example, found HeLa-derived human papillomavirus 18 (H-HPV18) in several of The Cancer Genome Atlas (TCGA) RNA-sequencing samples. This work motivated us to assess a greater number of samples and determine the origin of possible contaminations using viral sequences. To detect viruses with high specificity, we developed the publicly available workflow, VirDetect, that detects virus and laboratory vector sequences in RNA-seq samples. We applied VirDetect to 9143 RNA-seq samples sequenced at one TCGA sequencing center (28/33 cancer types) over 5?years. RESULTS:We confirmed that H-HPV18 was present in many samples and determined that viral transcripts from H-HPV18 significantly co-occurred with those from xenotropic mouse leukemia virus-related virus (XMRV). Using laboratory metadata and viral transcription, we determined that the likely contaminant was a pool of cell lines known as the "common reference", which was sequenced alongside TCGA RNA-seq samples as a control to monitor quality across technology transitions (i.e. microarray to GAII to HiSeq), and to link RNA-seq to previous generation microarrays that standardly used the "common reference". One of the cell lines in the pool was a laboratory isolate of MCF-7, which we discovered was infected with XMRV; another constituent of the pool was likely HeLa cells. CONCLUSIONS:Altogether, this indicates a multi-step contamination process. First, MCF-7 was infected with an XMRV. Second, this infected cell line was added to a pool of cell lines, which contained HeLa. Finally, RNA from this pool of cell lines contaminated several TCGA tumor samples most-likely during library construction. Thus, these human tumors with H-HPV or XMRV reads were likely not infected with H-HPV 18 or XMRV.

SUBMITTER: Selitsky SR

PROVIDER: S-EPMC6986043 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Virus expression detection reveals RNA-sequencing contamination in TCGA.

Selitsky Sara R SR Marron David D Hollern Daniel D Mose Lisle E LE Hoadley Katherine A KA Jones Corbin C Parker Joel S JS Dittmer Dirk P DP Perou Charles M CM

BMC genomics 20200128 1

<h4>Background</h4>Contamination of reagents and cross contamination across samples is a long-recognized issue in molecular biology laboratories. While often innocuous, contamination can lead to inaccurate results. Cantalupo et al., for example, found HeLa-derived human papillomavirus 18 (H-HPV18) in several of The Cancer Genome Atlas (TCGA) RNA-sequencing samples. This work motivated us to assess a greater number of samples and determine the origin of possible contaminations using viral sequenc ...[more]

PMID: 31992194

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Project description:Up to 20% of cancers worldwide are thought to be associated with microbial pathogens, including bacteria and viruses. The widely used methods of viral infection detection are usually limited to a few a priori suspected viruses in one cancer type. To our knowledge, there have not been many broad screening approaches to address this problem more comprehensively.In this study, we performed a comprehensive screening for viruses in nine common cancers using a multistep computational approach. Tumor transcriptome and genome sequencing data were available from The Cancer Genome Atlas (TCGA). Nine hundred fifty eight primary tumors in nine common cancers with poor prognosis were screened against a non-redundant database of virus sequences. DNA sequences from normal matched tissue specimens were used as controls to test whether each virus is associated with tumors.We identified human papilloma virus type 18 (HPV-18) and four human herpes viruses (HHV) types 4, 5, 6B, and 8, also known as EBV, CMV, roseola virus, and KSHV, in colon, rectal, and stomach adenocarcinomas. In total, 59% of screened gastrointestinal adenocarcinomas (GIA) were positive for at least one virus: 26% for EBV, 21% for CMV, 7% for HHV-6B, and 20% for HPV-18. Over 20% of tumors were co-infected with multiple viruses. Two viruses (EBV and CMV) were statistically significantly associated with colorectal cancers when compared to the matched healthy tissues from the same individuals (p?=?0.02 and 0.03, respectively). HPV-18 was not detected in DNA, and thus, no association testing was possible. Nevertheless, HPV-18 expression patterns suggest viral integration in the host genome, consistent with the potentially oncogenic nature of HPV-18 in colorectal adenocarcinomas. The estimated counts of viral copies were below one per cell for all identified viruses and approached the detection limit.Our comprehensive screening for viruses in multiple cancer types using next-generation sequencing data clearly demonstrates the presence of viral sequences in GIA. EBV, CMV, and HPV-18 are potentially causal for GIA, although their oncogenic role is yet to be established.

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Virus expression detection reveals RNA-sequencing contamination in TCGA.

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Virus expression detection reveals RNA-sequencing contamination in TCGA.

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