Abstract
Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called ‘Single Virus Genomics’, which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA). The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.
Highlights
Whole genome amplification and sequencing of single microbial cells is a powerful new tool in the field of microbial genomics, enabling direct examination of the genomic contents of individual cells without the need of cultivation [1,2,3]
For this study, flow cytometry was used to sort a mixed viral assemblage consisting of two known viruses; E. coli bacteriophages lambda and T4
A subsequent experiment to quantify virus particles within agarose droplets using Confocal Laser Scanning Microscopy (CLSM) indicated that 75% contained 1 or .1 (1–5), viruses (Table S2); and amplification of genomic material via multiple displacement amplification (MDA) was successful in 92% of virus-containing droplets
Summary
Whole genome amplification and sequencing of single microbial cells is a powerful new tool in the field of microbial genomics, enabling direct examination of the genomic contents of individual cells without the need of cultivation [1,2,3]. There is a higher likelihood of nonspecific DNAs preferentially amplified due to the lower quantity of template viral DNA as opposed to single bacterial cells as a result of the significant difference in particle (cell) size and genomic DNA content (25–100 nm; ,1.5femtograms for viruses, as opposed to 0.2–1.5 um; ,14femtograms for bacteria). To address this potential shortcoming the incubation time of genome amplification was reduced and we took advantage of the massively parallel, high-throughput capabilities of pyrosequencing to ensure both adequate coverage of the lambda genome and to examine the nature of any nonspecific amplification. Testing new enzyme and reagent lots prior to use and the reduction of free DNA through nuclease treatment should help to reduce nonspecific amplification
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