Abstract
The most efficient method to determine the genomic sequence of a dsDNA phage is to use a whole genome shotgun approach (WGSA). Preparation of a library where each genomic fragment has an equal chance of being represented is critical to the success of the WGSA. For many phages, there are regions of the genome likely to be under-represented in the shotgun library, which results in more gaps in the shotgun assembly than predicted by the Poisson distribution. However, as phage genomes are relatively small, this increased number of gaps does not present an insurmountable impediment to using the WGSA. This chapter will focus on construction of a high-quality random library and sequence analysis of this library in a 96-well format. Techniques are described for the mechanical fragmentation of genomic DNA into 2 kb average size fragments, preparation of the fragmented DNA for shotgun cloning, and advice on the choice of cloning vector for library preparation. Protocols for deepwell block culture, plasmid isolation, and sequencing in 96-well format are given. The rationale for determining the total number of random clones from a library to sequence for a 50 and 150 kb genome is explained. The steps involved in going from hundreds of shotgun sequencing traces to generating contigs will be outlined as well as how to close gaps in the sequence by primer walking on phage DNA and PCR-generated templates. Finally, examples will be given of how biological information about the phage genomic termini can be derived by analysis of the organization of individual clones in the shotgun sequence assembly. Specific examples are given for the circularly permuted termini of pac type phages, the direct terminal repeats found in most T7-like phages, variable host DNA at either end as in the Mu-like phages, and the \(5^{\prime}\) and \(3^{\prime}\) overhanging ends of cos type phages. The end result of these steps is the entire DNA sequence of a novel phage, ready for gene prediction.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Brussow H, Hendrix RW. Phage genomics: small is beautiful. Cell 2002;108(1):13–6.
Hendrix RW. Bacteriophage genomics. Curr.Opin.Microbiol. 2003;6(5):506–11.
Ackermann HW. Bacteriophage observations and evolution. Res.Microbiol. 2003; 154(4):245–51.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989.
Summer EJ, Gonzalez CF, Boomer M, Carlile T, Embry A, Kucherka AM et al. Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex. J.Bacteriol. 2006;188(1):255–68.
Summer EJ, Gonzalez CF, Carlisle T, Mebane LM, Cass AM, Savva CG et al. Burkholderia cenocepacia phage BcepMu and a family of Mu-like phages encoding potential pathogenesis factors. J.Mol.Biol. 2004;340(1):49–65.
Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res. 1998;8(3):195–202.
Casjens SR, Gilcrease EB, Winn-Stapley DA, Schicklmaier P, Schmieger H, Pedulla ML et al. The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy. J. Bacteriol. 2005;187(3):1091–104.
Thomas CA, Jr., MacHattie LA. Circular T2 DNA molecules. Proc.Natl.Acad.Sci.U.S.A 1964;52:1297–301.
Scholl D, Kieleczawa J, Kemp P, Rush J, Richardson CC, Merril C et al. Genomic analysis of bacteriophages SP6 and K1-5, an estranged subgroup of the T7 supergroup. J.Mol.Biol. 2004;335(5):1151–71.
Port E, Sun F, Martin D, Waterman MS. Genomic mapping by end-characterized random clones: a mathematical analysis. Genomics 1995;26(1):84–100.
Lander ES, Waterman MS. Genomic mapping by fingerprinting random clones: a mathematical analysis. Genomics 1988;2(3):231–9.
Wang J, Jiang Y, Vincent M, Sun Y, Yu H, Wang J et al. Complete genome sequence of bacteriophage T5. Virology 2005;332(1): 45–65.
Oefner PJ, Hunicke-Smith SP, Chiang L, Dietrich F, Mulligan J, Davis RW. Efficient random subcloning of DNA sheared in a recirculating point-sink flow system. Nucleic Acids Res. 1996;24(20):3879–86.
Roe B.A., Crabtree JS, Khan AS. DNA Isolation and Sequencing. John Wiley & Sons; 1996.
Anderson S. Shotgun DNA sequencing using cloned DNase I-generated fragments. Nucleic Acids Res. 1981;9(13):3015–27.
Boyle JS, Lew AM. An inexpensive alternative to glassmilk for DNA purification. Trends Genet. 1995;11(1):8.
Tautz D, Renz M. An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal.Biochem. 1983;132(1):14–9.
Inoue H, Nojima H, Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene 1990;96(1):23–8.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Summer, E.J. (2009). Preparation of a Phage DNA Fragment Library for Whole Genome Shotgun Sequencing. In: Clokie, M.R., Kropinski, A.M. (eds) Bacteriophages. Methods in Molecular Biology™, vol 502. Humana Press. https://doi.org/10.1007/978-1-60327-565-1_4
Download citation
DOI: https://doi.org/10.1007/978-1-60327-565-1_4
Publisher Name: Humana Press
Print ISBN: 978-1-60327-564-4
Online ISBN: 978-1-60327-565-1
eBook Packages: Springer Protocols