Microbial Oceanography Course

The project description is the Moore-supported Microbial Oceanography Course run out of Bermuda Institute of Ocean Sciences with sequencing run by PSU. PI is Craig A. Carlson and Co-PIs are Craig E. Nelson, Steven Giovannoni, and John Heidelberg.

here is a summary of how the sequences were trimmed, cleaned, and dereplicated to provide only unique sequences:

First low quality pyrosequencing reads were removed (average q-value <25, homopolymers >10bp, length <280bp or any ambiguous base), the forward sequencing primer was trimmed (338r; TGCWGCCWCCCGTAGGWGT), and sequences were assigned to samples according to multiplex barcodes excluding all sequences without an exact barcode and CA checksum linker sequence match. In order to remove non-unique sequences, chimeras, and other PCR artifacts, all sequences were aligned against the most recent SILVA database (v104; Pruesse et al. 2007) and ends were trimmed to a common length. From this alignment, poorly aligned sequences were excluded, chimeras were identified and removed using Chimera Slayer (courtesy of Brian Haas at the Broad Institute), and the alignment was further dereplicated to remove all non-unique sequences allowing for 2 bp differences in alignment according to Huse, et al (2010). All processing steps were conducted within the mothur bioinformatic software environment (Schloss 2009).

Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, et al. (2007). SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Research 35:7188 -7196.

Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, et al. (2009). Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities. Applied and Environmental Microbiology 75:7537-7541.

Haas, B. Microbiome Utilities Portal of the Broad Institute. http://microbiomeutil.sourceforge.net/ (Accessed December 9, 2010).

Huse SM, Welch DM, Morrison HG, Sogin ML. (2010). Ironing out the wrinkles in the rare biosphere through improved OTU clusteringemi_2193 1889.. 1898. Environmental Microbiology 12:1889?1898.