DNA was extracted from 16 samples using the E.Z.N.A. Stool DNA Kit (Omega Bio-tek, Norcross, GA, USA). The DNA extracted from stool samples was used to construct Illumina paired-end libraries following Illumina’s genomic DNA library preparation procedure. Then, the amplicon library was paired-end–sequenced (2 by 150) on an Illumina HiSeq X platform. Raw FASTQ files were quality-filtered using Trimmomatic (version 0.36) with the following criteria: (i) removal of bases at the start and end of a read below a threshold quality (score < 3) and (ii) the reads were truncated at any site receiving an average quality score of <15 over a 4-bp sliding window, discarding the truncated reads that were shorter than 100 bp.

BMTagger (version 3.102) was used to remove host contamination following the standard operating protocol for removing human sequence, as outlined in the Human Microbiome Project. Read level functional profiling with KO (KEGG Ortholog) was performed using the UProC toolbox (version 2.0.0-rc1) (37). The KEGG BRITE databases were used for annotation at the higher functional level. Briefly, the reads from the same KO were binned to REACTION ontology using the KO and REACTION hierarchy relationship, and unique reads were summarized to construct the REACTION ontology count table.

Metaphlan2 (version 2.5.0) and Kaiju (version 1.4.5) with default databases were used to perform the taxonomy composition analysis (38). The raw reads count table for several different taxonomy levels from Kaiju classifier was constructed for differential abundance analysis using DESeq2. R package DESeq2 (version 1.10.1) was used to detect the differential abundance ontologies or taxonomy catalog (39). The de bruijn graph–based assembler MEGAHIT (version 1.0.6) was used to assemble short reads. The contigs with length greater than 500 bp were used to make the reference index for bowtie alignment, the unaligned reads were collected to co-assemble with the same assembly parameter, and the minimal contig length was set to 200. MetaProdigal (version 2.6.3) was used for gene prediction of metagenomic contigs (40). The abundance of each nonredundant gene [measured as transcripts-per-million (TPM)] was quantified with kallisto (version 0.42.5). The function space of the nonredundant gene was annotated with KEGG.

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