Sensitive Detection of Viral Transcripts in Human Tumor Transcriptomes

Abstract

In excess of % of human cancer incidents have a viral cofactor. Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered. Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts. However, technical problems such as low abundances of viral transcripts in large volumes of sequencing data, viral sequence divergence, and homology between viral and human factors significantly confound identification of tumor viruses. We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors. We apply the approach to conducting the first systematic search for viruses in neuroblastoma, the most common cancer in infancy. The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor. However, a viral etiology of neuroblastoma is currently contested. We mapped transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses. Analysis of controls, comparisons with related methods, and statistical estimates demonstrate the high sensitivity of our approach. Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses. Likewise, de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens. While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates analyzed. Therefore, our results suggest that frequent viral cofactors of metastatic neuroblastoma are unlikely.

Lack of human cytomegalovirus expression in single cells from glioblastoma tumors and cell lines

“…Most of this debate stems from the fact that different research groups, often using different assays, have generated evidence to support or refute the presence of HCMV in GBM tumors. There is a large amount of research supporting both sides of this issue (Amirian, Bondy, Mo, Bainbridge, & Scheurer, 2014; Baryawno et al, 2011; Baumgarten et al, 2014; Cosset et al, 2014; Fornara et al, 2016; Hashida et al, 2015; Holdhoff et al, 2015; Joseph, McDermott, Baryshnikova, Cobbs, & Ulasov, 2017; Khoury et al, 2013; Lehrer, Green, Rosenzweig, & Rendo, 2015; Libard et al, 2014; McFaline-Figueroa & Wen, 2017; Prins, Cloughesy, & Liau, 2008; Rahbar et al, 2012; Sardi et al, 2015; Schelhorn et al, 2013; Shamran et al, 2015; Solomon, Ramkissoon, Milner, & Folkerth, 2014; Stangherlin et al, 2016; Strong et al, 2016; Wolmer-Solberg et al, 2013) that we summarize in Fig. 1B–D.…”

Tumour virology in the era of high-throughput genomics

“…Variability in sequencing depth is typically accounted for by normalizing to the total number of obtained reads, for example by stating viral expression levels as ‘reads per kilobase and million base pairs sequenced’ (RPKM) or in parts-per-million (ppm) of total library reads. Greater sensitivity for detecting highly diverged viral strains or new viruses can be obtained by first assembling non-human sequences into longer contiguous segments (contigs), followed by searches for homology to known viral reference sequences ( figure 1 b ) [ 7 , 9 , 12 , 13 , 15 , 16 , 18 ]. Furthermore, sites of viral genomic integration can be bioinformatically pinpointed by identification of discordant paired reads or chimeric human-viral sequences ( figure 1 c , discussed below).…”

The complete genome sequence of a novel virus, bellflower veinal mottle virus, suggests the existence of a new genus within the family Potyviridae

“…In brief, a transcriptome library was generated using an Illumina TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA), and de novo assembly of the quality-filtered NGS reads was performed using the Trinity pipeline. Assembled contigs were analyzed by BLASTn and BLASTx searches against the viral reference genome database in GenBank [5]. The entire procedure of RNA sequencing was performed by Macrogen Inc. (Seoul, South Korea).…”