Patterns in microbialite genetic composition relate to environmental variables
In preparation for pyrosequencing on a Genome Sequencer FLX Titanium 454 Life Sciences System (Roche 454 Life Sciences, Cd USA), a second round of PCR was performed using fusion primers that incorporated a variable length sample-specific nucleotide sequence (“barcodes”), and the 454 pyrosequencing adaptor A onto the 5’ end of the TX9 primer, and adaptor B onto the 5’ end of the 1391R primer. The barcodes enabled multiplexing several samples into a single 454 sequencing run, after PAGE purifying and quantitating the amplicons. The multiplexed amplicon library was sequenced using the standard 454 Life Sciences Lib-L emulsion PCR protocol and Titanium chemistry sequencing (Margulies et al., 2005). Raw sequence reads were quality filtered using the default 454 parameters as well as post instrument filters, that required all bases be Q20 or higher and the average of all bases in any read to be Q25 or greater. Sequences were trimmed to remove the TX9 primer from the 5’ end and at a conserved site distal to V6 on the 3’ (ca position 1067, E. coli numbering), resulting in an approximately 250 basepair read covering the V5 and V6 regions. Finally, to avoid diversity overestimation of the rare biosphere only sequences occurring at least 10 times were included in subsequent analysis. This approach has been proposed to reduce pyrosequencing noise after careful examination of whole genomes sequenced by the Sanger method and pyrosequencing with 454 technology (Hamp et al. 2009). The genetic data were organized in a file with 10 rows corresponding to the study localities and 5284 columns representing the different sequences obtained. The data are frequencies of each sequence. Sequences shared between the microbialites and the water column controls were eliminated from the analysis.  Both runs have all 20 samples (and therefore all 20 barcodes) present
UNAM
Microbialite diversity