Characterization of gut microbiome
The adhesion and colonization of the microbiota inside fish guts may be influenced by several factors linked to the stomach, pyloric caeca, and intestine portions (Ringø et al., 2003). Thus, we evaluated the microbiome in both the anterior and posterior portion of the guppy intestinal tracts. We extracted DNA from anterior and posterior sections (250 x 2 = 500 total) following the manufacturer’s protocol of the MoBio PowerSoil DNA Extraction kit. We submitted DNA to the Michigan State University Core Genomics Facility for Illumina sequence library construction using the Illumina TruSeq Nano DNA Library Preparation Kit and sequencing. We submitted a total of 508 samples for sequencing, randomized across two lanes. Eight samples (four anterior and four posterior) were included in both lanes to serve as controls. Following the core facility’s standard protocols, bacterial 16S V4 (515f/806r) Illumina compatible libraries were prepared using primers containing both the target sequences and the dual indexed Illumina compatible adapters (see Kozich et al. 2013). Completed libraries were normalized using Invitrogen SequalPrep DNA Normalization plates, pooled and cleaned up using AmpureXP magnetic beads. One sample failed to amplify so 507 samples were sequenced. 16S amplicon pools were sequenced independently in a 2x250bp paired end format using independent v2 500 cycle Illumina MiSeq reagent cartridges (Illumina, CA, USA).
Bacterial and fungal bioinformatics pipelines are fully described in Appendix S1. Briefly, reads were quality filtered and merged using the USEARCH pipeline (http://drive5.com/usearch/). Primers and adapter bases were removed using cutadapt (Martin 2011). Bacterial reads were filtered and truncated to 250 bp, clustered into operational taxonomic units (OTUs) at 97% identity level then classified against SILVAv123 rRNA database (Yilmaz et al. 2014). Sequences were aligned and a phylogenetic tree was built using PASTA (Mirarab et al. 2015). We removed (i) OTUs classified to Chloroplast and Mitochondria, (i ) OTUs present in less than 5 (of 507) samples, (iii ) OTUs that had less than 20 reads across all samples, and (iv ) samples with less than 2000 reads (Table S1). For calculating diversity metrics, we rarefied samples to 8000 reads. Bioinformatic code is publicly available (https://gitlab.msu.edu/belldere/guppy_gut_2015).