Shotgun metagenomic library was constructed from fecal DNA with the Nextera DNA sample preparation kit (Illumina, San Diego, CA), as per manufacturer’s specification. Barcoding indices were inserted using Nextera indexing kit (Illumina). Products were purified using Agencourt AMpure XP kit (Beckman Coulter, Brea, CA) and pooled for sequencing. Samples were sequenced using MiSeq reagent kit V2 (Illumina) in a HiSeq2500 sequencer.
Raw sequences were sorted using assigned barcodes and cleaned up before analysis (barcodes removed and sequences above a quality score, Q ≥ 30 taken forward for analyses). For assembly and annotation of sequences, MetAMOS47 (link) pipeline or Partek Flow software (Partek® Flow®, Partek Inc., St. Louis, MO) were used. These softwares provide powerful tools to filter unique hits between human and mouse-specific genes versus microbial signatures. Alpha and Beta diversity calculations were done using embedded programs within the metagenomic pipeline, or using Stata15 (StataCorp LLC, College Station, TX) or EXPLICET software.48 (link) Functional profiling was performed using HUMAnN2-0.11.149 (link) with Uniref50 database to implement KEGG orthologies.
Raw sequences were sorted using assigned barcodes and cleaned up before analysis (barcodes removed and sequences above a quality score, Q ≥ 30 taken forward for analyses). For assembly and annotation of sequences, MetAMOS47 (link) pipeline or Partek Flow software (Partek® Flow®, Partek Inc., St. Louis, MO) were used. These softwares provide powerful tools to filter unique hits between human and mouse-specific genes versus microbial signatures. Alpha and Beta diversity calculations were done using embedded programs within the metagenomic pipeline, or using Stata15 (StataCorp LLC, College Station, TX) or EXPLICET software.48 (link) Functional profiling was performed using HUMAnN2-0.11.149 (link) with Uniref50 database to implement KEGG orthologies.
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