Storr's Lake Microbial Mat Partial 16S rRNA gene sequencing-Pinnacle mound
The microbial community composition of the microbialites in Storr’s Lake was determined by using a barcoded 16S rRNA gene analysis. Genomic DNA (UltraClean™ Soil DNA Isolation Kit, MoBio Laboratories, Carlsbad, CA, USA) was extracted in triplicate from microbial mats associated with the calcerous knobs, plateau, mushroom and pinnacle mound microbialites, as well as a nonlithifying cheesecake microbial mat. The extracted DNA was shipped to MoGene, LC (St. Louis, MO, USA) for further analysis. MoGene quantified the DNA by using a Quant-iT Picogreen dsDNA Assay kit (Invitrogen, Carlsbad, CA) and PCR-amplified using barcoded primers targeting the V1-V3 region of the 16S rRNA gene. The bacterial primers used were the 27-Forward AGRGTTTGATCMTGGCTCAG (Weisburg et al., 1991) and 518-Reverse CGTATTACCGCGGCTGCTGG (Muyzer et al., 1993). The primers incorporated the Titanium Lib-A adaptor sequences and barcode sequences specific for the individual samples (See below).   The PCR mixture included 1X Master mix (MOLZYM Mastermix 16S Basic, Molzym, Bremen, Germany), 0.2 µM each of the MID forward and reverse primers, 0.51 U Taq DNA polymerase (MolTaq, Molzym), 30 ng of the template DNA, and DNA-free water for a final volume of 25 µL. Conditions for PCR were 96°C for 5 min, 25 cycles of 95°C for 10 sec, 56°C for 30 sec and 72°C for 30 sec, followed by a single cycle of 72°C for 5 min. The amplicons were then purified with a 1.8% agarose gel purification run (QIAquick Gel Extraction kit, Qiagen, Chatsworth, CA). The purified amplicons were examined with an Agilent Bioanalyzer DNA 7500 (Agilent, Santa Clara, CA) to determine concentration and assess the size of the products. The amplicon libraries were normalized, pooled and then pyrosequenced with Titanium chemistry (454 GS-FLX, Roche, Branford, CT, USA). The bioinformatics software program QIIME was used for processing, cluster analysis, and classification of the raw sequences (Caporaso et al., 2010). Following is the sequence pattern for the different samples: Sample-Standard Adaptor A Sequence-Key-Barcode-16S rDNA position 27 Forward Primer (Pinnacle Mound-CCATCTCATCCCTGCGTGTCTCCGAC-TCAG-TCGCACTAGT-AGRGTTTGATCMTGGCTCAG). The reverse primer has the following components:Standard Adaptor B Sequence (Reverse)-Key-Barcode-16S rDNA position 518 Reverse Primer (Reverse Complement):CCTATCCCCTGTGTGCCTTGGCAGTC-TCAG-N/A-CGTATTACCGCGGCTGCTGG.