Metagenomic Library Preparation, Sequencing and Quality Assessment

Metagenomic shotgun sequencing libraries were prepared and sequencing at CINVESTAV-LANGEBIO, Irapuato, Mexico. For each sample ∼5 μg of genomic DNA (DO 260/280 ≤1.8) was used with Illumina’s TruSeq nano for library preparation, which supports shearing by Covaris ultrasonication. Fragments were selected according to agarose gel 0.7% at 70 mV in order to obtain an average insert size of 550 bp. Libraries were sequenced using the Illumina MiSeq Paired-End 2 × 300 bp technology with a run in a single plate generating 12Gb of sequence data for all 12 samples. Quality of raw reads was analyzed using FastQC v0.11 (Andrews, 2010). TruSeq Indexed Adapter and barcodes were removed using cutadapt v1.12 (Martin, 2011). Low quality sequences were discarded with Trimmomatic using a sliding window of 4 bp, an average quality per base of 20, and min read length of 36 bp (Bolger et al., 2014). The assembly of the trimmed reads was conducted with Megahit v1.1.1-2 using the option – presets meta-large (Li et al., 2014).

The coding regions were searched from the obtained contigs using Prodigal v2.6.3 (Hyatt et al., 2010) with the -a option, to obtain the translated amino acid sequences of the predicted coding regions and -p meta option. The peptide amino acid sequences were then scanned against Pfam-A v30 (Finn et al., 2015). The abundance profile of each Pfam domain in the metagenomic samples was obtained from a Perl script extract_pfam.pl which is part of the MEBS software suite (De Anda et al., 2017). The resulting FASTA files of sequence contigs have been deposited in the MG-RAST repository under project number mgp80319.