GSE157610
Differential Peptide Subtraction (DPS):  mass spectrometry for human cancer virus discovery
The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host-derived molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass-spectrometry (dMS) followed by targeted analysis to facilitate this discovery. To trace the non-human dMS identified peptides back to its genetic origin by next generation sequencing (NGS) cDNA libraries were generated using degenerate oligos corresponding to the identified peptides. In a pilot study DPS identified both viral and human biomarkers. Based on the identified peptides that are differentially expressed in the virus positive tumor sample, degenerate oligos were designed. Degenerate oligos or LNA modified degenerate oligos or a modified oligo(dT) SMART primer were used to facilitate reverse transcription from viral or viral and host RNA. NGS analysis revealed seven times more MCV reads in degenerate oligo primed RNAseq samples compared to polyA-based sequencing reads. Overall design: Ribodepleted RNA from Merkel cell polyomavirus (MCV) positive Merkel cell carcinoma (MCC) tissue was used for RNA sequencing. Three different cDNA libraries were prepared using the same tissue RNA. Sample 1 (MCC-deg): a pool of degenerate oligos used to prime the cDNA synthesis. Sample 2 (MCC-LNAdeg): a pool of LNA modified degenerate oligos were used for cDNA synthesis. Sample 3 (MCC-polyA): modified oligo dT primer was used for cDNA synthesis prior to library generation. RNAseq data was analyzed to detect virus specific reads and counts were compared to evaluate the efficiency of library genertion.