16S multiplexed sequences of Soil and Litter bacteria, bacterial fire feedbacks
We extracted DNA from 0.25g of each 2g subsample. We used MoBio PowerSoil Kits (MoBio, Carlsbad, USA), then quantified DNA concentration of extracts using Qubit 2.0 (LifeTechnologies, Carlsbad, USA). The V4 hyper-variable region of 16S rDNA was amplified from 5ng of template DNA using the standard Earth Microbiome Primers 515F and 806R (Caporaso et al., 2011) and Q5 polymerase (New England Biosystems, Ipswich, USA). PCR protocol consisted of an initial denaturing at 98C for 30 seconds; then 25 cycles of denaturing at 98C for 10 seconds, annealing at 55C for 30 seconds, and extension at 72C for 30 seconds; followed by a final extension at 72C for 5 minutes. PCR products were cleaned using Agencourt AMPure XP magnetic beads (Beckman Coulter, Indianapolis, USA). A second PCR was then used to ligate unique Nexterra indices (Illumina, San Diego, USA) to each sample. Cycling conditions for the indexing PCR were similar to the first PCR, except for only running for 8 cycles, and then purified as before. Individual libraries were pooled at equimolar concentrations into a single, 4mM library; concentration and amplicon size was verified using a TapeStation 2200 (Agilent, Santa Clara, USA). Amplicon sequences were then generated using a 301bp, paired-end run on an Illumina Mi-seq (Illumina, San Diego, USA) at the Kansas State Integrated Genomics Center. Sequencing data included a negative water-only library control (from 1st PCR step above) and 120 samples, generated from soil and litter in the 60 plots. Paired reads were concatenated, and Nexterra indices were used as barcodes for demultiplexing the sample reads from the 120 sequenced samples.
multiplexed-library