protocols & resources

Helpful protocols and resources.

Protocols

Choosing the most appropriate protocol for preparing your sample for Roche/454 pyrosequencing ensures the highest quality data in the shortest amount of time.

Eukaryotic cDNA

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:-.

Bacterial cDNA

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.

Amplicon barcoding

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.

Improved Amplicon sequencing protocol

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

Software

We like QIIME (pronounced chime).

Roche Protocols

Amplicon Fusion Primer Design Guidelines for GS FLX Titanium Series Lib-A Chemistry

364k PDF

TCB-11002_Short Fragment Removal Procedure

318k PDF

Titanium chemistry Rapid Library Preparation Method Manual

1,903k PDF

Titanium chemistry Amplicon Library Preparation Method Manual

1,003k PDF

Titanium chemistry cDNA Rapid Library Preparation Method Manual

1,197k PDF

Amplicon (PCR Product) Sequencing Tips APP No. 001-2010

310k PDF

Guidelines for amplicon experimental design

953k PDF

Articles

A few representative publications that have either been published by customers or staff of engencore.

High-Throughput Sequencing and Metagenomics

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.

A core gut microbiome in obese and lean twins

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?

The influence of sex, handedness, and washing on the diversity of hand surface bacteria

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.

The Marine Viromes of Four Oceanic Regions

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

423k PDF, opens in a new browser window

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.