title: microbial community of Dastychius improvisus
design: Next generation sequencing of V3-V4 hypervariable regions in the bacterial 16S rRNA gene was performed using the following universal bacterial primer set: 341F (5-CCTACGGGNGGCWGCAG-3) and 785R (5-GACTACHVGGGTATCTAATCC-3) (Eiler et al. 2012). The library was prepared with a two-step PCR protocol based on Illuminas 16S metagenomic library prep guide using Q5 HotStart High-Fidelity DNA Polymerase (NEBNext) and the Nextera Index kit (2250bp) according to the manufacturers protocol. Pooled amplicons were sequenced using a MiSeq sequencer, and a paired-end (PE) technology using Illumina v2 putty (Genomed, Poland), following the manufacturers run protocols (Illumina, Inc., San Diego, CA, USA). Automatic de-multiplexing of raw reads and primary sequences analysis were performed on a MiSeq machine using the MiSeq Reporter (MSR) v. 2.6 (16S Metagenomics Protocol). Next-generation reads were subsequently clustered into operational taxonomic units (OTUs) at 97% similarity by using the uclust method, v. 1.2.22q125 (Edgar 2010).