Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

Barzon, Luisa; Lavezzo, Enrico; Militello, Valentina; Toppo, Stefano; Palù, Giorgio

Computational subtraction analysis of data obtained using conventional shotgun sequencing methods has been used to identify viral sequences (e.g., HBV, HCMV, human papillomaviruses 18 and 16, HHV8, HCV, EBV and human spumavirus) in EST libraries derived from normal and cancerous tissues [40] and in post-transplant lymphoproliferative disorder tissue [41] . In these studies, computational subtraction analysis relied on sequence data gathered for other purposes as the yield of viral sequences was very low due to the predominance of human sequences. However, exploiting the great amount of sequencing data achievable by NGS methods, computational subtraction analysis could become a method of choice for viral discovery. This approach has been used for the discovery of a new polyomavirus associated with most cases of Merkel cell carcinoma (MCC) [42] . MCC is a rare and aggressive human skin cancer that typically affects elderly and immunosuppressed individuals, a feature which was suggestive of an infectious origin. RNA was purified from MCC samples and analyzed by 454 pyrosequencing. Digital transcriptome subtraction of all human sequences led to the detection of a fusion transcript between a human receptor tyrosine phosphatase and a Large T antigen sequence related to murine polyomaviruses. This sequence was used as starting point for whole genome sequencing and characterization of this previously unknown polyomavirus that was called Merkel cell polyomavirus (MCPyV). The presence of the virus in 80% MCC tissues but only in about 10% of control tissues from various body sites, including the skin, and the demonstration that, in MCPyV-positive MCCs, viral DNA was integrated within the tumor genome in a clonal pattern, strongly suggested the etiological role of the virus in the pathogenesis of MCC [42] .