title: 16s survey of salt marsh enrichment
design: The V3-V4 hypervariable regions of SSU rRNA gene were PCR amplified and the amplicons were sequenced on the Illumina MiSeq platform by FISABIO, Valencia, Spain (http://fisabio.san.gva.es/en/servicios). Library preparations were performed as described previously (51). The 16S rRNA gene V3-V4 variable region was amplified using PCR primers 341F/785R S-D-Bact-0341-b-S-17, 5-CCTACGGGNGGCWGCAG-3 (52), and S-D-Bact655 0785-a-A-21, 5-GACTACHVGGGTATCTAATCC-3 (52,53). The raw data generated this way were further filtered (direct removal of barcodes and primer sequences, sequences shorter than 150 bp, as well as sequences with ambiguous base calls and with homopolymer runs longer than 6 bp) by the same sequencing service. Pre-processed sequences were analysed with the NGS analysis pipeline of the SILVA rRNA gene database project (SILVAngs 1.3) 660 (54). Each sequence was aligned using the SILVA Incremental Aligner (SINA v1.2.10) for ARB SVN (55) against the SILVA SSU rRNA SEED and quality-controlled database (54). After quality control steps, sequences were de-replicated on a per-sample basis. Identical reads were collapsed and the unique reads were clustered (OTUs 98% similarity) using cdhit-est (version 3.1.2; http://www. bioinformatics.org/cd-hit) (56). The reference read of each 665 OTU was classified by a local nucleotide BLAST search against the non-redundant version of the SILVA SSU Ref dataset (release 132, Dec 13, 2017; http://www.arb-silva.de) using Blastn (version 2.2.30+; http://blast.ncbi.nlm.nih.gov/Blast.cgi) with default settings