Sequencing viral genomes from a single isolated plaque

BackgroundWhole genome sequencing of viruses and bacteriophages is often hindered because of the need for large quantities of genomic material. A method is described that combines single plaque sequencing with an optimization of Sequence Independent Single Primer Amplification (SISPA). This method can be used for de novo whole genome next-generation sequencing of any cultivable virus without the need for large-scale production of viral stocks or viral purification using centrifugal techniques.MethodsA single viral plaque of a variant of the 2009 pandemic H1N1 human Influenza A virus was isolated and amplified using the optimized SISPA protocol. The sensitivity of the SISPA protocol presented here was tested with bacteriophage F_HA0480sp/Pa1651 DNA. The amplified products were sequenced with 454 and Illumina HiSeq platforms. Mapping and de novo assemblies were performed to analyze the quality of data produced from this optimized method.ResultsAnalysis of the sequence data demonstrated that from a single viral plaque of Influenza A, a mapping assembly with 3590-fold average coverage representing 100% of the genome could be produced. The de novo assembled data produced contigs with 30-fold average sequence coverage, representing 96.5% of the genome. Using only 10 pg of starting DNA from bacteriophage F_HA0480sp/Pa1651 in the SISPA protocol resulted in sequencing data that gave a mapping assembly with 3488-fold average sequence coverage, representing 99.9% of the reference and a de novo assembly with 45-fold average sequence coverage, representing 98.1% of the genome.ConclusionsThe optimized SISPA protocol presented here produces amplified product that when sequenced will give high quality data that can be used for de novo assembly. The protocol requires only a single viral plaque or as little as 10 pg of DNA template, which will facilitate rapid identification of viruses during an outbreak and viruses that are difficult to propagate.

[1]  A. Djikeng,et al.  Viral genome sequencing by random priming methods , 2008 .

[2]  Alejandro A. Schäffer,et al.  A Fast and Symmetric DUST Implementation to Mask Low-Complexity DNA Sequences , 2006, J. Comput. Biol..

[3]  G. Church,et al.  Sequencing genomes from single cells by polymerase cloning , 2006, Nature Biotechnology.

[4]  B. Andresen,et al.  Genomic analysis of uncultured marine viral communities , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[5]  M. Cáccamo,et al.  A Viral Discovery Methodology for Clinical Biopsy Samples Utilising Massively Parallel Next Generation Sequencing , 2011, PloS one.

[6]  P. Salamon,et al.  Metagenomic Analyses of an Uncultured Viral Community from Human Feces , 2003, Journal of bacteriology.

[7]  A. Djikeng,et al.  Metagenomic Analysis of RNA Viruses in a Fresh Water Lake , 2009, PloS one.

[8]  Marcel Martin.  Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[9]  Phillip I. Tarr,et al.  Metagenomic Analysis of Human Diarrhea: Viral Detection and Discovery , 2008, PLoS pathogens.

[10]  K. Paithankar,et al.  Precipitation of DNA by polyethylene glycol and ethanol. , 1991, Nucleic acids research.

[11]  B. O’Malley,et al.  Fractionation of chick oviduct chromatin. Nuclease-resistant deoxyribonucleic acid. , 1975, Biochemical Journal.

[12]  C. Hutchison,et al.  Cell-free cloning using φ29 DNA polymerase , 2005 .

[13]  J. Lis.  Fractionation of DNA fragments by polyethylene glycol induced precipitation. , 1980, Methods in enzymology.

[14]  D. Fouts.  Phage_Finder: Automated identification and classification of prophage regions in complete bacterial genome sequences , 2006, Nucleic acids research.

[15]  C. Hutchison,et al.  Cell-free cloning using phi29 DNA polymerase. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[16]  R. Blakesley,et al.  Linear amplification DNA sequencing directly from single phage plaques and bacterial colonies. , 1991, Nucleic acids research.

[17]  A. Gnirke,et al.  High-quality draft assemblies of mammalian genomes from massively parallel sequence data , 2010, Proceedings of the National Academy of Sciences.

[18]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[19]  G. Reyes,et al.  Sequence-independent, single-primer amplification (SISPA) of complex DNA populations. , 1991, Molecular and cellular probes.

[20]  T. Czerny.  High primer concentration improves PCR amplification from random pools. , 1996, Nucleic acids research.

[21]  Roger S Lasken,et al.  Unbiased whole-genome amplification directly from clinical samples. , 2003, Genome research.

[22]  Tao Zhang,et al.  RNA Viral Community in Human Feces: Prevalence of Plant Pathogenic Viruses , 2005, PLoS biology.

[23]  Raul Andino,et al.  Quasispecies Theory and the Behavior of RNA Viruses , 2010, PLoS pathogens.

[24]  D. Spiro,et al.  PB2 Residue 158 Is a Pathogenic Determinant of Pandemic H1N1 and H5 Influenza A Viruses in Mice , 2010, Journal of Virology.

[25]  S. Frackman,et al.  Betaine and DMSO : Enhancing Agents for PCR , 2003 .