Faecal genomic DNA was extracted using a combination of mechanical and chemical lysis via FastPrep Instrument (MP Biomedicals) and QIAamp Fast DNA Stool Mini Kit (Qiagen) as described previously [18]. The V3-V6 region on prokaryotic 16S rRNA was amplified from total DNA as previously described [7376]. Amplicons were sheared using the Covaris LE220 sonicator (Covaris, Inc., USA) and built into sequencing libraries using GeneRead DNA Library I Core Kit (Qiagen) according to the manufacturer’s protocol. DNA libraries were multiplexed by 96 indices, pooled, and sequenced on the Illumina HiSeq 2500 using paired-end (2x76bp) sequencing. Sequencing reads were demultiplexed (Illumina bcl2fastq software) and filtered (PF = 0) before conversion to FASTQ format.

The average numbers of sequencing quality-passed reads mapping to the 16S rRNA Greengenes global rRNA database (dated May 2013; greengenes/13_5/99_otus.fasta) are described in the Supplementary Table S2.

Read trimming from 3′ was done to remove bases with quality score ≤ 2, followed by additional removal of reads pairs shorter than 60 bp after trimming [7377]. Following read trimming, full length 16S rRNA (V3-V6 region) reconstructions were produced from the short sequencing reads using the EMIRGE amplicon (Expectation Maximisation Iterative Reconstruction of Genes from the Environment) algorithm [7578]. The analysis method is described in the publication of Ong and colleagues [73] and the updated tools and pipeline are available at: https://github.com/CSB5/GERMS_16S_pipeline. EMIRGE leverages 16S rRNA sequences on the SILVA database for template guided assembly of reconstructions of the 16S rRNA amplicon sequences [77, 78]. EMIRGE prevents chimeric sequences from mapping. The reconstructed 16S rRNA sequences (at least 99% sequence similarity) are collapsed into OTUs, and Graphmap [79] was used to map these OTUs to the Greengenes global rRNA database (dated May, 2013; greengenes/13_5/99_otus.fasta) [80]. OTUs were called at various taxonomic levels of identity (species, genus, family etc.) [81]. EMIRGE assigns abundance estimates to the reconstructed 16S rRNA sequences. The relative abundance of OTUs was determined for each sample and converted to relative abundances at various taxonomic level (phylum, family, genus, species level).

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