Gut Microbiome Profiling via 16S rRNA Sequencing

DNA was extracted using a Microbial DNA extraction kit (QIAGEN)^{70 (link)} and 50 µl from a capsule device or 100 mg stool. The 16S rRNA amplicons were generated using Earth Microbiome Project-recommended 515F/806R primer pairs and 5PRIME HotMasterMix (Quantabio, 2200410) with the following program in a thermocycler: 94 °C for 3 min, 35 cycles of 94 °C for 45 s, 50 °C for 60 s and 72 °C for 90 s, followed by 72 °C for 10 min. PCR products were cleaned, quantified and pooled using the UltraClean 96 PCR Cleanup kit (QIAGEN, 12596-4) and Quant-iT dsDNA High Sensitivity Assay kit (Invitrogen, Q33120). Samples were sequenced with 300-bp reads on a MiSeq (Illumina). Sequence data were de-multiplexed using the Illumina bcl2fastq algorithm at the Chan Zuckerberg BioHub Sequencing facility. Subsequent processing was performed using the R statistical computing environment v.4.0.3 (ref. ⁷¹ ) and DADA2 as previously described using pseudo-pooling^{72 (link)}. truncLenF and truncLenR parameters were set to 250 and 180, respectively. Taxonomy was assigned using the Silva rRNA database v.132 (ref. ^{73 (link)}). Samples with >2,500 reads were retained for analyses.