Hydrogen-isotopic variability in fatty acids from Yellowstone National Park hot spring microbial communities
DNA was extracted from samples using the Powersoil extraction kit (MoBio, Carlsbad, CA). The manufacturer's protocol was followed with the 10 min lysis/vortexing step replaced by 1 min bead-beating. qPCR was conducted on a Lightcycler 480 II (Roche, Indianapolis, IN) to monitor the plateau point of amplification. A PCR-touchdown annealing temperature strategy (Don, 2010) was employed for 10 cycles to minimize primer dimer formation and the final 15-20 cycles combined the annealing and elongation steps. PCR was stopped for each sample when it appeared the amplification was beginning to plateau. qPCR volumes were 30 ï­L total, 9.6 ï­L of which was DNA template. Reagents and final concentrations were: 1X Phusion DNA Polymerase MasterMix with HF Buffer (New England Biolabs, Ipswich, MA), 0.5 ï­M forward and reverse primer, 8% v/v DMSO, Sybr Green I. Primers incorporated the adapter sequences for pyrosequencing on the GSFLX platform of the Roche 454 Pyrosequencing technology. Additionally, each forward primer had an 8 nt barcode corresponding to an environmental sample that allowed amplicon pools to be sequenced in parallel and binned by sample in silico post-sequencing. The SSU rRNA gene primers were attached to adapters only (reverse primer) or adapters and barcodes (forward primer) by a 2 nt linker. The sequence for the linker was the two least abundant bases at the adjacent positions to the primer in the Silva SSU_Ref_102_NR alignment and database (Pruesse, 2007) for which the E.coli sequence did not have a gap. SSU rRNA gene primers were adapted from 515F (Lane, 1991) and 927R (Jurgens et al., 1997) to account for observed mis-pairings of the primers with archaeal and bacterial sequences from the Silva SSU_Ref_102_NR database. Evaluation of primers was done using custom Python scripts that employed bioinformatics modules from PyCogent (Knight, 2007). The specific SSU rRNA gene primers used were 515f-modified, 5'GTGYCAGCMGCCGCGGTAA3', and 927r-modified, 5'CCGYCAATTCMTTTRAGTTT3'. Individual sample amplicon volumes were pooled and gel purified (Montage DNA Gel Extraction Kit. Millipore, Billerica, MA) prior to sequencing. Although, GSFLX adapters were used during PCR amplification, sequencing was done on the later generation Roche 454 Titanium platform.