Miseq reagent kit v3 v4

Total bacterial DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) following the manufacturer’s instructions. The library formation and sequencing of the 16S rRNA gene were performed with MiSeq® Reagent Kit V3-V4 on MiSeq-Illumina® platform. Generated sequences were analysed using the subsampled open reference operational taxonomic unit (OTU) strategy with default settings in QIIME (v1.9.1). The paired-end reads were merged and demultiplexed. Subsampled open-reference OTU-picking was performed using UCLUST with 97% sequence similarity. Representative sequences were chimera checked using Blast_fragments approach with default settings and taxonomy assigned against the Greengenes database V13_8 using the UCLUST method with a 90% confidence threshold. The singletons and OTUs with relative abundance across all samples below 0.005% were excluded as recommended by Bokulich et al. [26 (link)]. The samples that reported low sequencing yield (less than 5,000 reads after quality check) were excluded from the analysis.

The collection of fecal samples was conducted by participants under the guidance of trained doctors within a 2-day period. On the first day, the doctor explained how to collect fecal samples for all the participants and distributed sampling tubes to the participants. On the second day, the fecal samples were examined and collected by the trained doctors before blood collection and anthropometric measurements. A commercial analysis kit (OMEGA Mag-bind universal pathogen 96 kit) was used to extract DNA following the manufacturer’s instructions. V3–V4 hypervariable regions of the bacteria 16s rDNA were then amplified. The purified amplicons were fluorescently quantified and further sequenced on the Illumina Miseq high-throughput sequencing platform with Miseq reagent kit V3-V4 and PhiX control V3–V4.