16S rRNA gene sequencing of Sphagnum microbiome
Sphagnum individuals were collected from the field and immediately frozen at -20C until processing. Each individual was crushed to a powder in liquid N2, and DNA was extracted from ~100 mg of ground sample using the DNeasy Plant Pro Kit (QIAGEN, Valencia, CA) according to the manufacturers instructions. Amplification of the 16S rRNA gene V4 region was performed using the primers 515F-Y (5-GTGYCAGCMGCCGCGGTAA) and 806R-Apprill (5-GGACTACNVGGGTWTCTAAT) primers. . Each primer was modified to include an Illumina overhang adapter sequence on the 5 end for subsequent barcoding. Reactions were performed in triplicate using of 2.5-ng DNA template in a solution containing 1X HotStarTaq Master Mix (QIAGEN), 0.4 mg/ml bovine serum albumin (BSA), 0.2 M of each primer, and 0.76 M of each mitochondrial (mPNA) and plastid (pPNA) peptide nucleic acid (PNA) clamps. PNA clamps have been shown to reduce plant plastid and mitochondrial DNA amplification in PCR reactions. Triplicate PCR products were pooled together and sequenced on an Illumina MiSeq2000 platform using a 500-cycle v2 sequencing kit (250 paired-end reads) at the Georgia Tech High Throughput DNA Sequencing Core in Atlanta, GA. Before submitting to the SRA, the primer and sequencing adapters were removed using Cutadapt software.