Name: Lisa Zeigler

Email: lzeigler@jcvi.org

Author: Lisa A. Zeigler, and Shannon J. Williamson

Author affiliation: J. Craig Venter Institute, Scripps Institution of Oceanography

Abstract title: Single Virus Genomics: Bridging the Gap Between Cultivation and Metagenomics One Virus at a Time

Absstract:

Whole genome amplification and sequencing of single microbial cells has revolutionized microbial ecology by allowing researchers to directly examine the genomic contents of individual cells in the absence of prior cultivation efforts. The field of environmental viral metagenomics has gained momentum over the past five years, but sequencing of environmental viral genomes is still dependent on the establishment of cultivable virus-host systems. To circumvent the potential bias from pre-selective factors associated with culturing techniques and increase genomic DNA recovery, we have designed a novel systematic approach that extends single cell amplification to single viral particles, thereby creating a link between isolation and characterization of individual viral particles and whole-community metagenomic analyses. To accomplish this task, we have adapted flow-cytometric methods for the capture of individual viral particles into an agarose matrix. As proof-of-concept, we captured Enterobacteriophage T4 and lambda viral particles into a single agarose droplet on a microscope slide. The viral particles were visualized on the slide using epifluorscence and confocal microscopy to show a single viral particle was present in each droplet of agarose. Viruses were lysed, and then their genomic material was amplified using phi29 DNA polymerase and the multiple displacement amplification (MDA) reaction. The amplified viral genomes were subsequently screened for genes specific to T4 (gp23 major head protein) and lambda (integrase) through PCR. The individual viral genomes were then sequenced using Sanger technology to validate single particle isolation. We are now extending this technique to natural virioplankton communities where we have preformed metagenomics analyses. Using fingerprinting techniques, we plan to classify isolates based on their genotypes prior to sequencing in order to cover the diversity apparent in these communities. These results will then be used to select varied genomic isolates for further sequence analyses to increase our knowledge of the whole-genome diversity among virioplankton communitites.