Analysis of high-throughput sequencing and annotation strategies for phage genomes

Matthew R. Henn, Matthew B. Sullivan, Nicole Stange-Thomann, Marcia S. Osburne, Aaron M. Berlin, Libusha Kelly, Chandri Yandava, Chinnappa Kodira, Qiandong Zeng, Michael Weiand, Todd Sparrow, Sakina Saif, Georgia Giannoukos, Sarah K. Young, Chad Nusbaum, Bruce W. Birren, Sallie W. Chisholm

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

Background: Bacterial viruses (phages) play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. Methodology/Principal Findings: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles), and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL) or of a whole genome shotgun library (WGSL), or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. Conclusions/Significance: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.

Original languageEnglish (US)
Article numbere9083
JournalPLoS One
Volume5
Issue number2
DOIs
StatePublished - Feb 5 2010
Externally publishedYes

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Bacteriophages
bacteriophages
Genes
Throughput
Genome
genome
genomics
DNA
Firearms
DNA Sequence Analysis
Horizontal Gene Transfer
Gene transfer
Informatics
Genomic Library
methodology
Public Sector
Genomics
Open Reading Frames
Libraries
Ecosystem

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Henn, M. R., Sullivan, M. B., Stange-Thomann, N., Osburne, M. S., Berlin, A. M., Kelly, L., ... Chisholm, S. W. (2010). Analysis of high-throughput sequencing and annotation strategies for phage genomes. PLoS One, 5(2), [e9083]. https://doi.org/10.1371/journal.pone.0009083

Analysis of high-throughput sequencing and annotation strategies for phage genomes. / Henn, Matthew R.; Sullivan, Matthew B.; Stange-Thomann, Nicole; Osburne, Marcia S.; Berlin, Aaron M.; Kelly, Libusha; Yandava, Chandri; Kodira, Chinnappa; Zeng, Qiandong; Weiand, Michael; Sparrow, Todd; Saif, Sakina; Giannoukos, Georgia; Young, Sarah K.; Nusbaum, Chad; Birren, Bruce W.; Chisholm, Sallie W.

In: PLoS One, Vol. 5, No. 2, e9083, 05.02.2010.

Research output: Contribution to journalArticle

Henn, MR, Sullivan, MB, Stange-Thomann, N, Osburne, MS, Berlin, AM, Kelly, L, Yandava, C, Kodira, C, Zeng, Q, Weiand, M, Sparrow, T, Saif, S, Giannoukos, G, Young, SK, Nusbaum, C, Birren, BW & Chisholm, SW 2010, 'Analysis of high-throughput sequencing and annotation strategies for phage genomes', PLoS One, vol. 5, no. 2, e9083. https://doi.org/10.1371/journal.pone.0009083
Henn MR, Sullivan MB, Stange-Thomann N, Osburne MS, Berlin AM, Kelly L et al. Analysis of high-throughput sequencing and annotation strategies for phage genomes. PLoS One. 2010 Feb 5;5(2). e9083. https://doi.org/10.1371/journal.pone.0009083
Henn, Matthew R. ; Sullivan, Matthew B. ; Stange-Thomann, Nicole ; Osburne, Marcia S. ; Berlin, Aaron M. ; Kelly, Libusha ; Yandava, Chandri ; Kodira, Chinnappa ; Zeng, Qiandong ; Weiand, Michael ; Sparrow, Todd ; Saif, Sakina ; Giannoukos, Georgia ; Young, Sarah K. ; Nusbaum, Chad ; Birren, Bruce W. ; Chisholm, Sallie W. / Analysis of high-throughput sequencing and annotation strategies for phage genomes. In: PLoS One. 2010 ; Vol. 5, No. 2.
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