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...
| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Public Library of Science
2010
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| Online Access: | http://hdl.handle.net/1721.1/54796 |
| _version_ | 1826192756120223744 |
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| author | Chisholm, Sallie (Penny) Sullivan, Matthew B. Birren, Bruce W. Nusbaum, Chad Young, Sarah K. Giannoukos, Georgia Saif, akina Sparrow, Todd Weiand, Michael Zeng, Qiandong Kodira, Chinnappa D. Yandava, Chandri Kelly, Libusha Berlin, Aaron M. Osburne, Marcia Stange-Thomann, Nicole Henn, Matthew R. |
| author2 | Massachusetts Institute of Technology. Department of Civil and Environmental Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Civil and Environmental Engineering Chisholm, Sallie (Penny) Sullivan, Matthew B. Birren, Bruce W. Nusbaum, Chad Young, Sarah K. Giannoukos, Georgia Saif, akina Sparrow, Todd Weiand, Michael Zeng, Qiandong Kodira, Chinnappa D. Yandava, Chandri Kelly, Libusha Berlin, Aaron M. Osburne, Marcia Stange-Thomann, Nicole Henn, Matthew R. |
| author_sort | Chisholm, Sallie (Penny) |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T09:28:26Z |
| format | Article |
| id | mit-1721.1/54796 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T09:28:26Z |
| publishDate | 2010 |
| publisher | Public Library of Science |
| record_format | dspace |
| spelling | mit-1721.1/547962022-09-30T14:38:27Z Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes Chisholm, Sallie (Penny) Sullivan, Matthew B. Birren, Bruce W. Nusbaum, Chad Young, Sarah K. Giannoukos, Georgia Saif, akina Sparrow, Todd Weiand, Michael Zeng, Qiandong Kodira, Chinnappa D. Yandava, Chandri Kelly, Libusha Berlin, Aaron M. Osburne, Marcia Stange-Thomann, Nicole Henn, Matthew R. Massachusetts Institute of Technology. Department of Civil and Environmental Engineering Chisholm, Sallie (Penny) Chisholm, Sallie (Penny) Sullivan, Matthew B. 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. 2010-05-14T19:35:14Z 2010-05-14T19:35:14Z 2010-02 2010-01 Article http://purl.org/eprint/type/JournalArticle 1932-6203 http://hdl.handle.net/1721.1/54796 Henn MR, Sullivan MB, Stange-Thomann N, Osburne MS, Berlin AM, et al. (2010) Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes. PLoS ONE 5(2): e9083. doi:10.1371/journal.pone.0009083 en_US http://dx.doi.org/10.1371/journal.pone.0009083 PLoS ONE Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Public Library of Science PLoS |
| spellingShingle | Chisholm, Sallie (Penny) Sullivan, Matthew B. Birren, Bruce W. Nusbaum, Chad Young, Sarah K. Giannoukos, Georgia Saif, akina Sparrow, Todd Weiand, Michael Zeng, Qiandong Kodira, Chinnappa D. Yandava, Chandri Kelly, Libusha Berlin, Aaron M. Osburne, Marcia Stange-Thomann, Nicole Henn, Matthew R. Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes |
| title | Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes |
| title_full | Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes |
| title_fullStr | Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes |
| title_full_unstemmed | Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes |
| title_short | Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes |
| title_sort | analysis of high throughput sequencing and annotation strategies for phage genomes |
| url | http://hdl.handle.net/1721.1/54796 |
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