The polycyclic aromatic hydrocarbon degradation potential of coastal sand microbial communities in the Gulf of Mexico
Custom fusion primers for PCR, consisting of an Illumina adaptor, 12 different inline barcodes (forward primer) or 8 different dedicated indices (reverse primer) were used to target and amplify the conserved regions of the V6 16S rRNA gene sequence for bacteria. For each library, PCR was carried out in triplicate 33 microliter reaction volumes with an amplification cocktail consisting of 1.0 U Platinum Taq Hi-Fidelity Polymerase, 1X Hi-Fidelity buffer, 200 uM dNTP PurePeak DNA polymerase mix, 1.5 mM MgSO4, and 0.2 micromolar of each primer.  10-25 ng template DNA was added to each PCR reaction and a no-template control was added for each primer pair.  Cycling conditions were as follows: 94C for 3 min denaturation, then 30 cycles of 94C for 30s, 60C for 60s, and 72C for 90s, followed by a final 10 min extension at 72C.  The triplicate PCR reactions were pooled after amplification and were purified with a Qiaquick PCR 96-well PCR plate cleanup.  Purified DNA was eluted in 30 uL of Qiagen buffer EB.  PicoGreen quantification of the PCR products was used to determine the addition of equimolar amounts of each reaction. The final multiplexed pool was then size selected by a Pippin Prep and quantified using KapaBiosystems qPCR system before sequencing on a lane of an Illumina HiSeq 100 cycle paired-end run.  A combination of CASAVA 1.8.2 to identify reads by index and custom python scripts were used to demultiplex the datasets.  100% consensus between the overlapping regions of the paired-end sequence reads was used to eliminate the majority of sequencing errors.  For more details on the methods used see Eren et al., June 2013, PLOS ONE 8(6): e66643. doi:10.1371/journal.pone.0066643.g001