Sequalprep normalization plate

Fecal samples collected from participants were stored at 4 °C until delivered to the laboratory within a 36-h period. Anaerocult was used in order to preserve anaerobic species present within a sample. The samples were homogenized, aliquoted, and stored at − 80 °C until used for microbial DNA extraction. DNA extraction was performed using the Qiagen Powersoil kit with the addition of the heating step from the Powerfecal kit (heating at 65 °C for 10 min). Resulting microbial DNA was subjected to PCR in order to amplify the V4 region of the 16S rRNA gene. The resulting library fragments were normalized using the SequalPrep normalization plates (Invitrogen, Carlsbad, CA). The libraries were pooled and analyzed via an Agilent Bioanalyzer trace. Samples were split into two sequencing runs in order to increase sample read depth. Samples were grouped together by family groups (twins, spouses) in order to make sure all samples from a family were sequenced in the same sequencing run. Sequencing data was generated on the MiSeq platform, using a 2 × 251 paired-end sequencing run with 20% Phix to increase base diversity during the run.

Microbial nucleic acids were extracted from a total of 192 fecal and 180 hide samples collected across the 12-week study via the MO BIO PowerViral DNA/RNA Isolation Kit™. After measuring DNA concentration with the Qubit® Fluorometer, the V3-V4 region of the 16S rRNA gene was amplified using dual-indexed primers [28 ], with an annealing temperature of 52 °C for 32 cycles. Aliquots of amplicon reactions (3 μL) were electrophoresed on agarose gels to verify amplification. Amplicons were normalized with SequalPrep™ Normalization Plates (Invitrogen Inc., CA, USA), pooled, and then sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA) using the paired-end 250 base pair sequencing protocol. Sequence data discussed in this publication have been uploaded to the DNA Databank of Japan under DDBJ BioProject accession number PRJDB4262.

SSU rRNA genes were amplified from gradient fractions (n = 20 per gradient) and from non-fractionated DNA from soil. Barcoded primers consisted of: 454-specific adapter B, a 10 bp barcode, a 2 bp linker (5′-CA-3′), and 806R primer for reverse primer (BA806R); and 454-specific adapter A, a 2 bp linker (5′-TC-3′), and 515F primer for forward primer (BA515F). Each PCR contained 1.25 U μl−1 AmpliTaq Gold (Life Technologies, Grand Island, NY; N8080243), 1X Buffer II (100 mM Tris-HCl, 500 mM KCl, pH 8.3), 2.5 mM MgCl2, 200 μM of each dNTP, 0.5 mg ml⁻¹ BSA, 0.2 μM BA515F, 0.2 μM BA806R, and 10 μL of 1:30 DNA template in 25 μl total volume. The PCR conditions were 95°C for 5min followed by 22 cycles of 95°C for 10 s, 53°C for 30 s, and 72°C for 30 s, followed by a final elongation at 72°C for 5 min. Amplification products were checked by 1% agarose gel. Reactions were performed in triplicate and pooled. Amplified DNA was gel purified (1% low melt agarose) using the Wizard SV gel and PCR clean-up system (Promega, Madison, WI; A9281) per manufacturer's protocol. Samples were normalized by SequalPrep normalization plates (Invitrogen, Carlsbad, CA; A10510) or based on PicoGreen DNA quantification and pooled in equimolar concentration. Amplicons were sequenced on Roche 454 FLX system using titanium chemistry at Selah Genomics (Columbia, SC).