16S rRNA gene sequencing of soil biofilm
The V4-V5 region of the bacterial 16S ribosomal RNA gene was amplified by PCR (95  C for 2 min, followed by 25 cycles at 95  C for 30 s, 55  C for 30 s, and 72  C for 30 s and a final extension at 72  C for 5 min) using primers 515F 5 -barcode-GTGCCAGCMGCCGCGG)-3  and 907R 5 -CCGTCAATTCMTTTRAGTTT-3 , where barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate in a 20  L mixture containing 4  l of 5   FastPfu Buffer, 2  L of 2.5 mM dNTPs, 0.8  L of each primer (5  M), 0.4  l of FastPfu Polymerase, and 10 ng of template DNA. Amplicons were extracted from 2% agarose gels and purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer s instructions and were quantified using a QuantiFluor  -ST (Promega, U.S.). Purified amplicons were pooled to be equimolar and paired-end sequenced (2   250) on an Illumina MiSeq platform according to standard protocols.