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HeLa nucleic acid contamination in the cancer genome atlas leads to the misidentification of human papillomavirus 18.

ABSTRACT: We searched The Cancer Genome Atlas (TCGA) database for viruses by comparing non-human reads present in transcriptome sequencing (RNA-Seq) and whole-exome sequencing (WXS) data to viral sequence databases. Human papillomavirus 18 (HPV18) is an etiologic agent of cervical cancer, and as expected, we found robust expression of HPV18 genes in cervical cancer samples. In agreement with previous studies, we also found HPV18 transcripts in non-cervical cancer samples, including those from the colon, rectum, and normal kidney. However, in each of these cases, HPV18 gene expression was low, and single-nucleotide variants and positions of genomic alignments matched the integrated portion of HPV18 present in HeLa cells. Chimeric reads that match a known virus-cell junction of HPV18 integrated in HeLa cells were also present in some samples. We hypothesize that HPV18 sequences in these non-cervical samples are due to nucleic acid contamination from HeLa cells. This finding highlights the problems that contamination presents in computational virus detection pipelines.Viruses associated with cancer can be detected by searching tumor sequence databases. Several studies involving searches of the TCGA database have reported the presence of HPV18, a known cause of cervical cancer, in a small number of additional cancers, including those of the rectum, kidney, and colon. We have determined that the sequences related to HPV18 in non-cervical samples are due to nucleic acid contamination from HeLa cells. To our knowledge, this is the first report of the misidentification of viruses in next-generation sequencing data of tumors due to contamination with a cancer cell line. These results raise awareness of the difficulty of accurately identifying viruses in human sequence databases.

SUBMITTER: Cantalupo PG

PROVIDER: S-EPMC4442357 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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HeLa nucleic acid contamination in the cancer genome atlas leads to the misidentification of human papillomavirus 18.

Cantalupo Paul G PG Katz Joshua P JP Pipas James M JM

Journal of virology 20150128 8

<h4>Unlabelled</h4>We searched The Cancer Genome Atlas (TCGA) database for viruses by comparing non-human reads present in transcriptome sequencing (RNA-Seq) and whole-exome sequencing (WXS) data to viral sequence databases. Human papillomavirus 18 (HPV18) is an etiologic agent of cervical cancer, and as expected, we found robust expression of HPV18 genes in cervical cancer samples. In agreement with previous studies, we also found HPV18 transcripts in non-cervical cancer samples, including thos ...[more]

PMID: 25631090

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Project description:UnlabelledViruses are causally associated with a number of human malignancies. In this study, we sought to identify new virus-cancer associations by searching RNA sequencing data sets from >2,000 patients, encompassing 21 cancers from The Cancer Genome Atlas (TCGA), for the presence of viral sequences. In agreement with previous studies, we found human papillomavirus 16 (HPV16) and HPV18 in oropharyngeal cancer and hepatitis B and C viruses in liver cancer. Unexpectedly, however, we found HPV38, a cutaneous form of HPV associated with skin cancer, in 32 of 168 samples from endometrial cancer. In 12 of the HPV38-positive (HPV38(+)) samples, we observed at least one paired read that mapped to both human and HPV38 genomes, indicative of viral integration into the host DNA, something not previously demonstrated for HPV38. The expression levels of HPV38 transcripts were relatively low, and all 32 HPV38(+) samples belonged to the same experimental batch of 40 samples, whereas none of the other 128 endometrial carcinoma samples were HPV38(+), raising doubts about the significance of the HPV38 association. Moreover, the HPV38(+) samples contained the same 10 novel single nucleotide variations (SNVs), leading us to hypothesize that one patient was infected with this new isolate of HPV38, which was integrated into his/her genome and may have cross-contaminated other TCGA samples within batch 228. Based on our analysis, we propose guidelines to examine the batch effect, virus expression level, and SNVs as part of next-generation sequencing (NGS) data analysis for evaluating the significance of viral/pathogen sequences in clinical samples.ImportanceHigh-throughput RNA sequencing (RNA-Seq), followed by computational analysis, has vastly accelerated the identification of viral and other pathogenic sequences in clinical samples, but cross-contamination during the processing of the samples remain a major problem that can lead to erroneous conclusions. We found HPV38 sequences specifically present in RNA-Seq samples from endometrial cancer patients from TCGA, a virus not previously associated with this type of cancer. However, multiple lines of evidence suggest possible cross-contamination in these samples, which were processed together in the same batch. Despite this potential cross-contamination, our data indicate that we have detected a new isolate of HPV38 that appears to be integrated into the human genome. We also provide general guidelines for computational detection and interpretation of pathogen-disease associations.

Project description:The pandemic due to the outbreak of 2019 coronavirus disease (COVID-19) caused by novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised significant public health concerns. Rapid, affordable, and accurate diagnostic testing not only paves the way for the effective treatment of diseases, but also plays a crucial role in preventing the spreading of infectious diseases. Herein, a one-pot CRISPR/Cas13a-based visual biosensor was proposed and developed for the rapid and low-cost nucleic acid detection. By combining Cas13a cleavage and Recombinase Polymerase Amplification (RPA) in a one-pot reaction in a disposable tube-in-tube vessel, amplicon contamination could be completely avoided. The RPA reaction is carried out in the inner tube containing two hydrophobic holes at the bottom. After the completion of amplification reaction, the reaction solution enters the outer tube containing pre-stored Cas13a reagent under the action of centrifugation or shaking. Inner and outer tubes are combined to form an independent reaction pot to complete the nucleic acid detection without opening the lid. This newly developed nucleic acid detection method not only meets the need of rapid nucleic acid detection at home without the need for any specialized equipment, but also fulfils the requirement of rapid on-site nucleic acid detection with the aid of small automated instruments. In this study, CRISPR/Cas13a and CRISPR/Cas12a were used to verify the reliability of the developed one-pot nucleic acid detection method. The performance of the system was verified by detecting the DNA virus, i.e., African swine fever virus (ASFV) and the RNA virus, i.e., SARS-Cov-2. The results indicate that the proposed method possesses a limit of detection of 3 copy/μL. The negative and positive test results are consistent with the results of real-time fluorescence quantitative polymerase chain reaction (PCR), but the time required is shorter and the cost is lower. Thus, this study makes this method available in resource-limited areas for the purpose of large-scale screening and in case of epidemic outbreak.

Dataset Information

HeLa nucleic acid contamination in the cancer genome atlas leads to the misidentification of human papillomavirus 18.

Publications

HeLa nucleic acid contamination in the cancer genome atlas leads to the misidentification of human papillomavirus 18.

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