DPS - a rapid method for genome sequencing of DNA-containing bacteriophages directly from a single plaque.
Bacteriophages (phages) coexist with bacteria in all environments and influence microbial diversity, evolution and industrial production processes. As a result of this major impact of phages on microbes, tools that allow rapid characterization of phages are needed. Today, one of the most powerful methods for characterization of phages is determination of the whole genome using high throughput sequencing approaches. Here a direct plaque sequencing (DPS) is described, which is a rapid method that allows easy full genome sequencing of DNA-containing phages using the Nextera XT™ kit. A combination of host-DNA removal followed by purification and concentration of the viral DNA, allowed the construction of Illumina-compatible sequencing libraries using the Nextera™ XT technology directly from single phage plaques without any whole genome amplification step. This method was tested on three Caudovirales phages; ϕ29 Podoviridae, P113g Siphoviridae and T4 Myovirdae, which are representative of >96% of all known phages, and were sequenced using the Illumina MiSeq platform. Successful de novo assembly of the viral genomes was possible.
Department of Biology, Faculty of Science, University of Copenhagen, Universitetsparken 15, DK-2100 København Ø, Denmark. Electronic address: nws772@ku.dk.
Witold
W
Kot
Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark.
Finn K
FK
Vogensen
Department of Biology, Faculty of Science, University of Copenhagen, Universitetsparken 15, DK-2100 København Ø, Denmark.
Søren J
SJ
Sørensen
Department of Biology, Faculty of Science, University of Copenhagen, Universitetsparken 15, DK-2100 København Ø, Denmark; Department of Environmental Science, Aarhus University, Frederiksborgvej 399, Roskilde, Denmark.
Lars H
LH
Hansen
D001435:Bacteriophages / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D013997:Time Factors; D014773:Virology / Q000379:methods*
D004279:DNA, Viral
Full genome sequencing; High-throughput sequencing; Phages; Single plaque sequencing