Human infant gut 16S rRNA V3 region sequences: Sample 226.1
"DNA was extracted (PowerLyzer PowerSoil DNA isolation kit, MoBio 12855-100) from 250 mg feces according to the provided protocol with minor modifications: Bead beating was performed at 30 cycles/s for 10 min (Retsch MM 300 mixer mill) and the initial centrifugation steps were performed at 10,000 x g for 3 min, as recommended for clay matter. DNA quantity and quality was measured by Qubit dsDNA BR assay (InvitrogenTM, Q32850) and NanoDrop? 1000 (Thermo Scientific), respectively, yielding on average 33.3 ? 22.2 ng/microl DNA with A260/A280 = 1.82 ? 0.12 and A260/A230 = 1.61 ? 0.38. The PCR amplification of the V3-region of the 16S rRNA gene was performed with 5 ng community DNA as template, using 0.2 ?l Phusion High-Fidelity DNA polymerase (Fisher Scientific, F-553L), 4 ?l HF-buffer, 0.4 ?l dNTP (10 mM of each base),1 microM forward primer (PBU 5'A-adapter-TCAG-barcode-CCTACGGGAGGCAGCAG-3') and 1 microM reverse primer (PBR 5'-trP1-adapter-ATTACCGCGGCTGCTGG-3') in a 20 ?l total reaction volume. Both primers include sequencing adaptors and the forward primer additionally a unique 10-12 bp barcode (Ion Xpress Barcode Adapters). The PCR program included 30s at 98C, 24 cycles of 15s at 98C and 30s at 72C, followed by 5 min at 72C. The PCR product was purified using HighPrepTM PCR Magnetic Beads (MAGBIO, AC-60005) with the 96-well magnet stand (MAGBIO, MyMag 96), according to the prescribed procedure. DNA quantity was measured using Qubit dsDNA HS assay (InvitrogenTM, Q32851) and samples pooled to obtain equimolar libraries containing up to 90 samples in each library"
Barcode
CTGACATAATC
Cohort
SKOT I
Time point (age of infant)
9 months