Sequence-independent Amplification of Rotaviruses for High-throughput Next-generation Sequencing

Anthony K. Bennici1, Karla M. Stucker1, Asmik Akopov1, Nadia Fedorova1, Rebecca A. Halpin1, Timothy B. Stockwell2, David E. Wentworth
1Virology Group and 2Informatics Department, J. Craig Venter Institute, Rockville, MD 20850

Rotavirus is a segmented dsRNA virus that causes gastroenteritis and is the primary cause of severe pediatric diarrhea, which results in over half a million deaths per year globally. There are eight known species of rotaviruses (A-H) that infect humans and other animals, with Rotavirus A being the leading cause of human disease. Due to their great genetic diversity and broad public health implications, it is essential to increase existing genomic sequence data on rotaviruses. The goal of this project is to develop an in-house method for the sequence-independent amplification of viral dsRNA genomes that will be optimized for high-throughput viral genome sequencing using next generation sequencing platforms already in place at the J. Craig Venter Institute (JCVI). Sequence-independent amplification involves ligating a specialized adaptor to the dsRNA genome segments to allow for the synthesis of cDNA using an adaptor-specific primer. We will use a lab-adapted human strain of rotavirus with a known genome sequence to test and optimize this sequence-independent amplification method. Development of a high-throughput sequence-independent amplification strategy for dsRNA viruses will increase our ability to sequence more diverse rotavirus strains, including clinical isolates from both humans and animals, regardless of their nucleotide sequence diversity.