Open Access Method

A tool kit for quantifying eukaryotic rRNA gene sequences from human microbiome samples

Serena Dollive1, Gregory L Peterfreund1, Scott Sherrill-Mix1, Kyle Bittinger1, Rohini Sinha1, Christian Hoffmann1, Christopher S Nabel1, David A Hill123, David Artis123, Michael A Bachman45, Rebecca Custers-Allen1, Stephanie Grunberg1, Gary D Wu6, James D Lewis7 and Frederic D Bushman1*

Author Affiliations

1 Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, 3610 Hamilton Walk, Philadelphia, PA 19104, USA

2 Institute for Immunology, Perelman School of Medicine at the University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA 19104, USA

3 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA 19104, USA

4 Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, 3400 Spruce Street, Philadelphia, 19104, USA

5 Department of Pathology, University of Michigan, 1301 Catherine St, Ann Arbor, MI 48109, USA

6 Division of Gastroenterology, Perelman School of Medicine at the University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104, USA

7 Center for Clinical Epidemiology and Biostatistics, Perelman School of Medicine at the University of Pennsylvania, 423 Guardian Drive, Philadelphia, PA 19104, USA

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Genome Biology 2012, 13:R60  doi:10.1186/gb-2012-13-7-r60

Published: 3 July 2012

Additional files

Additional file 1:

Samples studied from known eukaryotic organisms.

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Additional file 2:

Comparison of PCR amplification reactions for DNA purified from stool using different methods. Average DNA yields were: PSP, 59.6 ng/μl; PowerSoil, 30.4 ng/μl; FastDNA extraction, 15.8 ng/μl; and the archaeal method, 12.7 ng/μl. PCR products were separated on an 0.8% agarose gel and stained with ethidium bromide. Top: amplification products generated using the 18S primer pair. Bottom: amplification products generated using the ITS1F-ITS2 primer pair.

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Additional file 3:

Samples studied from human stool.

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Additional file 4:

Analysis of DNA samples from known eukaryotes using BROCC, MARTA, and MEGAN. (a) 18S rRNA gene amplicons classified by all three classifiers. (b) ITS rRNA gene amplicons classified by all three classifiers. The sample tested is listed along the x-axis. Individual OTUs in each sample are shown by the points, which are sized in proportion to their read counts. A point is colored by the program and configuration used to classify that point. These data were classified by BROCC using default settings, MARTA using default settings, MARTA using a BLAST word size and voting thresholds to match the BROCC default settings, MEGAN using default settings and the same blastn output used by BROCC, and MEGAN using an abbreviated blastn output with a maximum of five hits per query sequence. The lowest level of correct classification for each OTU is listed on the y-axis.

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Additional file 5:

Comparison of BROCC, MARTA and MEGAN.

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Additional file 6:

Sequences of DNA oligonucleotides used in this study.

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Additional file 7:

Description of the BROCC program. (a) Pseudocode. (b) Flow chart of implementation.

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Additional file 8:

BROCC program parameters and options. Defaults were used in this study.

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Additional file 9:

BROCC source code. BROCC source code version 1.1.0.

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