Helpful protocols and resources.
Choosing the most appropriate protocol for preparing your sample for Roche/454 pyrosequencing ensures the highest quality data in the shortest amount of time.
Novaes, E., D. R. Drost, W. G. Farmerie, G. J. Pappas, D. Grattapaglia, R. R. Sederoff, and M. Kirst. 2008. High-throughput gene and SNP discovery in Eucalyptus grandis, an uncharacterized genome. Bmc Genomics 9:-.
Frias-Lopez, J., Y. Shi, G. W. Tyson, M. L. Coleman, S. C. Schuster, S. W. Chisholm, and E. F. DeLong. 2008. Microbial community gene expression in ocean surface waters. P Natl Acad Sci USA 105:3805-3810.
Sogin, M. L., H. G. Morrison, J. A. Huber, D. Mark Welch, S. M. Huse, P. R. Neal, J. M. Arrieta, and G. J. Herndl. 2006. Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proceedings of the National Academy of Sciences of the United States of America 103:12115-12120.
Full text available at PNAS In this article Noah Fierer describes an improved Amplicon sequencing protocol you might useful.
Sample isolation and cleanup
Virus, UltraClean Microcial DNA Isolation Kit, Roche 454 Pyrosequencing
Metagenomic Analysis of the Viral Communities in Fermented Foods
Eun-Jin Park, Kyoung-Ho Kim, Guy C. J. Abell, Min-Soo Kim, Seong Woon Roh, and Jin-Woo Bae. Appl. Envir. Microbiol., Feb 2011; 77: 1284 – 1291.
Bacteria, UltraClean Microbial DNA Isolation Kit, Pyrosequencing
Genome Sequence of a Novel Species, Propionibacterium humerusii
Susan M. Butler-Wu, Dhruba J. Sengupta, Weerayuth Kittichotirat, Frederick A. Matsen, III, and Roger E. Bumgarner J. Bacteriol., Jul 2011; 193: 3678
DNA, PowerClean DNA Clean-Up Kit, Pyrosequencing
Lachnospiraceae and Bacteroidales Alternative Fecal Indicators Reveal Chronic Human Sewage Contamination in an Urban Harbor
Ryan J. Newton, Jessica L. VandeWalle, Mark A. Borchardt, Marc H. Gorelick, and Sandra L. McLellan. Appl. Envir. Microbiol., Oct 2011; 77: 6972 – 6981.
Deep Sea Sediments, PowerMax DNA Isolation Kit, Genome Analyzer GAII instrument (Illumina)
Ultra-deep sequencing of foraminiferal microbarcodes unveils hidden richness of early monothalamous lineages in deep-sea sediments
Béatrice Lecroq, Franck Lejzerowicz, Dipankar Bachar, Richard Christen, Philippe Esling, Loïc Baerlocher, Magne Østerås, Laurent Farinelli, and Jan Pawlowski. PNAS, Aug 2011; 108: 13177 – 13182.
Symbiotic-fungus, PowerSoil DNA Isolation Kit, Pyrosequencing
Distinct Ectomycorrhizospheres Share Similar Bacterial Communities as Revealed by Pyrosequencing-Based Analysis of 16S rRNA Genes
S. Uroz, P. Oger, E. Morin, and P. Frey-Klett. Appl. Envir. Microbiol., Apr 2012; 78: 3020 – 3024.
Metal-Contaminated Stream Sediment, PowerSoil DNA Isolation Kit, GS 454 FLX Pyrosequencing
Mercury and Other Heavy Metals Influence Bacterial Community Structure in Contaminated Tennessee Streams
Tatiana A. Vishnivetskaya, Jennifer J. Mosher, Anthony V. Palumbo, Zamin K. Yang, Mircea Podar, Steven D. Brown, Scott C. Brooks, Baohua Gu, George R. Southworth, Meghan M. Drake, Craig C. Brandt, and Dwayne A. Elias, Appl. Envir. Microbiol., Jan 2011; 77: 302 – 311.
Skin Swabs, PowerSoil DNA Isolation Kit, Pyrosequencing
Forensic identification using skin bacterial communities
Noah Fierer, Christian L. Lauber, Nick Zhou, Daniel McDonald, Elizabeth K. Costello, and Rob Knight
PNAS, Apr 2010; 107: 6477 – 6481.
Oral swabs, PowerSoil DNA Isolation Kit, 454 Life Sciences FLX Sequencer
The bacterial microbiota in the oral mucosa of rural Amerindians Monica Contreras, Elizabeth K. Costello, Glida Hidalgo, Magda Magris, Rob Knight, and Maria G. Dominguez-Bello, Microbiology, Nov 2010; 156: 3282 – 3287.
Breast Tissue, FFPE DNA Isolation Kit, SOLiD Sequencing
Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens Yost SE, Smith EN, Schwab RB, Bao L, Jung H, Wang X, Voest E, Pierce JP, Messer K, Parker BA, Harismendy O, Frazer KA Nucleic Acids Res. 2012 Apr 6, 1-12
source: MO BIO Laboratories
We like QIIME (pronounced chime).
A few representative publications that have either been published by customers or staff of engencore.
265k PDF, opens in a new browser window
In this recent review paper in “Estuaries and Coasts”, Joe Jones summarizes some of the more salient issues that arise during preparation of samples for Roche/454 pyrosequencing.
Requires subscription to Nature
Is the hypothesis that there is a core human gut microbiome, definable by a set of abundant microbial organismal lineages that we all share incorrect?
Full text available at PNAS
Whose hands are dirtier, men or women? It turns out there are a lot more bacteria on our hands than previously thought as well as a greater diversity of bacteria between our two hands.
358k PDF, opens in a new browser window
The marine environment is a complex, poorly understood system. Viruses outnumber bacteria by several orders of magnitude but what controls their distribution and evolution?
Characterization of mutation spectra with ultra-deep pyrosequencing: Application to HIV-1 drug resistance
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HIV is not composed of a single strain. Instead it is composed of a variety of different subtypes- many of which are rare and have undetermined impacts of drug treatment. Pyrosequencing allows an in-depth look at both common and rare HIV strains thus allowing clinically relevant strains to be targeted.