The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
PLoS One. 2010 Feb 5;5(2):e9083. doi: 10.1371/journal.pone.0009083.
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.
细菌病毒（噬菌体）在塑造微生物种群方面发挥着关键作用，因为它们影响宿主死亡率和水平基因转移。因此，它们对当地和全球生态系统功能以及人类健康有着重大影响。尽管它们很重要，但人们对噬菌体所携带的基因组多样性知之甚少，因为捕获完整噬菌体基因组的方法受到目标基因组知识的缺乏以及为测序生成足够数量的基因组 DNA 的困难的阻碍。在目前公共领域中大约 550 个噬菌体基因组中，不到 5%是海洋噬菌体。
方法/主要发现：为了通过比较基因组方法推进噬菌体生物学的研究，我们使用海洋噬藻体作为模型系统。我们比较了 DNA 制备方法（直接从噬菌体裂解物或 CsCl 纯化的噬菌体颗粒中提取 DNA），以及利用 Sanger 测序连接扩增随机文库（LASL）或全基因组随机文库（WGSL）测序，或 454 焦磷酸测序方法的测序策略。我们证明了直接从噬菌体裂解物中制备基因组 DNA，结合 454 焦磷酸测序，最适合大规模噬菌体基因组测序，因为这种方法能够以高精度捕获完整的连续基因组。此外，我们描述了一种自动化注释信息学管道，该管道提供高质量的注释，在 ORF 调用中产生很少的假阳性和假阴性。
结论/意义：这些 DNA 制备、测序和注释策略为蓬勃发展的噬菌体基因组学领域提供了一种高通量方法。
