A novel clonal and ultrasensitive sequencing approach for studying the clinical significance of viral genetic variation in HIV infection

Changes in viral genetic variation within hosts alter the severity and spread of infectious diseases with important implications for clinical medicine and public health.

HIV infection is characterized by rapid and error-prone viral replication resulting in genetically diverse virus populations.

Understanding the rate of diversity and the mechanisms involved will provide important information to basic concepts of viral infections.

To characterize the development of viral diversity after infection, we propose to use a novel approach that accurately determines to the true viral population including the allele frequencies of the RNA population under study. Next generation sequencing offers the opportunity to view viral populations in radically new ways.

However, this technology suffers from introducing the problem of sequence resampling, recombination and by error rates of the sequencing platform.

Our preliminary data support the use of ultrasensitive single genome sequencing (uSGS) coupled to the Primer-ID approach, to track individual RNA molecules as a way to overcome the problems during library preparation. We will use this technology on a third generation sequencing platform (PacBio).

We will apply this combined approach to the study of viral populations to examine the role of genetic variation, epigenetic regulation and pre-existing drug resistance mutations as well as general features of population structure in HIV infection.