Metagenomic Profiling of Environmental Samples

Samples of 4 l were filtered using 0.22 μm Pall Supor filters (Pall Corporation, Port Washington, NY, USA). DNA extraction from filter was carried out using the RapidWater DNA extraction kit (MoBio, Carlsbad, CA, USA). Sequencing libraries were prepared using Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer instructions, and quality checked using Caliper LabChip GX. Sequencing was performed on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) with 100 bp paired-ends and an insert size of 250 bp. Raw sequencing reads were quality checked using fastq-mcf [45 ], and reads derived from human contamination were removed after mapping on the human genome (version hg19) using bowtie2 [46 (link)]. Adaptors and residual synthetic constructs were removed first mapping the reads on the genome sequence of the phage Phy174, and then using trim galore [47 ].
Microbiome profiling based on a database of taxon-specific marker genes was performed using MetaPhlAn2 [48 (link)]. The output of MetaPhlAn2 analyses was plotted using GraPhlAn [49 (link)].