Miseq 2 150 bp paired end platform

Fecal and intestinal microbiota DNA was extracted using the PowerLyzer PowerSoil DNA Isolation Kit (MoBio, Carlsbad CA) and the PowerSoil-htp 96 Well Soil DNA Isolation Kit (MoBio). The amplicon library of V4 regions of the bacterial 16S rRNA genes were obtained by triplicate PCR with barcoded fusion primers, quantification with the Qubbit 2.0 Fluorometer (Life Technologies, Carlsbad, CA), and combination of each DNA sample at equal concentrations as previously described (Livanos et al., 2016 (link)). The library was sequenced with the Ilumina MiSeq 2 × 150 bp paired end platform (Ilumina, San Diego CA) at the NYUMC Genome Technology Center.

Zhang et al. collected fecal samples from non-obese diabetic mice from Jackson Laboratory (4 (link)). Then, they extracted intestinal microbiota DNA using the PowerLyzer PowerSoil DNA isolation kit (MoBio, Carlsbad, CA) and the PowerSoil-htp 96-Well Soil DNA isolation kit (MoBio). Then, they acquired the amplicon library of V4 regions of the 16S rRNA genes through triplicate PCR with barcoded fusion primers, quantification with the Qubbit 2.0 Fluorometer (Life Technologies, Carlsbad, CA). and combination of each DNA sample at equal concentrations as in (2 (link)). Then, they sequenced the library using Illumina MiSeq 2 × 150 bp paired end platform (Illumina, San Diego, CA).
We used QIIME 2.0 [16] to obtain the feature table, taxonomic annotations, and phylogenetic tree. We retained only the reads with >75% of the original length while removing the reads with more than three consecutive low-quality bases (Phred score < 20). We constructed (1 (link)) the feature table using the 97% sequence similarity through open reference piking based on the Greengenes database (63 (link)), (2 (link)) the taxonomic annotations using the RDP classifier while removing chimeras using ChimeraSlayer (64 (link)), and (3 (link)) the phylogenetic tree using FastTree (65 (link)).