Amplicon of human: stool day 98
All samples were processed at the APC Microbiome Ireland laboratory (Cork, Ireland). DNA was extracted from 0.2g of a stool sample using the repeated bead-beating plus column method as described by Yu and Morrison (2004) and Ghosh et al. (2020) with minor modifications, including one extraction blank to monitor potential contamination. Nucleic acids were purified using the QIAmp Fast DNA Stool Mini Kit (Qiagen, UK), and DNA was stored at -20C. DNA concentration was quantified using a Qubit flurometer (Life Technologies Corp., Carlsbad, CA). Amplicon libraries were prepared according to the Illumina 16S Metagenomic Sequencing Library Preparation protocol, targeting the V3-V4 region of the 16S rRNA gene. Amplicon was performed using Illumina adapter-tailed primers: Forward: 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and Reverse: 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. The PCR amplification was carried out with KAP HiFi HotStart ReadyMix (Roche, Switzerland) using the following conditions: initial denaturation at 95C for 3 min; 25 cycles of 95C for 30 sec, 55C for 30 sec, and 72C for 30 sec; final extension at 72C for 5 min. A subsequent eight-cycle index PCR was performed to attach dual indices using the Nextera XT index kit v2 Set C (Illumina, San Diego, CA, USA). PCR products were visualized on a 1.5% agarose gel stained with SYBR safe /Thermo Fisher, MA, USA), purified with AMPure XP beads (Beckman Coulter, CA, USA), and quantified using the Qubit 1X dsDNA HS Assay Kit (Thermo Fisher). Fragment size distributions were confirmed on an Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Libraries were normalized to 20 nM, pooled in equal volumes (20 L per sample), and sequenced at Genewiz (Leipzig, Germany) on an Illumina MiSeq platform (2 x 300 bp paired-end reads).
SUB16038228
Omni_22