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


RESULTS
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
These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.
Henn
Matthew R
MR
The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America. mhenn@broadinstitute.org
Sullivan
Matthew B
MB
Stange-Thomann
Nicole
N
Osburne
Marcia S
MS
Berlin
Aaron M
AM
Kelly
Libusha
L
Yandava
Chandri
C
Kodira
Chinnappa
C
Zeng
Qiandong
Q
Weiand
Michael
M
Sparrow
Todd
T
Saif
Sakina
S
Giannoukos
Georgia
G
Young
Sarah K
SK
Nusbaum
Chad
C
Birren
Bruce W
BW
Chisholm
Sallie W
SW
D001435:Bacteriophages / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D016679:Genome, Viral / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D048929:Prochlorococcus / Q000821:virology; D017422:Sequence Analysis, DNA / Q000379:methods*
D004279:DNA, Viral; D003851:Deoxyribonucleases