Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

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

Investigation of retrovirus and retroviral vector integration sites in host cell chromosomes is another field of viral oncology which received a great contribution from NGS technologies. The use of viral vectors that integrate in host genome for gene transfer may cause malignant transformation due to activation of host proto-oncogenes or inactivation of tumor-suppressor genes, as a consequence of viral vector integration within these genes [48] [49] [50] . Deep sequencing technology has been used to map the integration sites of retroviruses and HIV [51] , as well as retroviral and HIV-based vectors for gene therapy and cell reprogramming [52] [53] [54] . Deep sequencing methods for detection of retrovirus integration are based on 454 pyrosequencing of products of ligation-mediated PCR (LM-PCR) [55, 56] or linear amplification-mediated PCR (LAM-PCR) [57] . Both LM-PCR and LAM-PCR use restriction enzymes to fragment the DNA of interest containing proviruses. Then, digested DNA is ligated with a compatible linker and amplified by PCR using primers that anneal in the LTR and in the linker sequence. Nested primers containing linkers for the 454 protocol are then used for a second PCR, which is processed by 454 high-throughput sequencing. A LAM-PCR method without the use of restriction enzymes was also developed for high throughput sequencing [58] . Recently, a new method was developed for recovering sites of integrated DNA based on the bacterial transposase MuA. The transposase is used to introduce adaptors into genomic DNA to allow PCR amplification and analysis by 454 pyrosequencing. This method could avoid the bias associated with restriction enzymes and recovered integration sites in a near random fashion. It provided a measure of cell clonal abundance, which is crucial for detecting expansion of cell clones that may be a prelude to malignant transformation [59] .