Infant stools were collected from diapers and aliquoted within 24 hours into sterile tubes and stored at −80° C. DNA extraction was then conducted using Zymo DNA extraction kits (Tustin, CA, USA) on thawed samples, followed by quantity and purity assessment using OD260/280 nanodrop measurement (19 (link)). Fusion primers were used to obtain 16S ribosomal RNA (rRNA) V4-V5 amplicons. One of eight five-nucleotide long forward primers was used to connect the Illumina-specific bridge and sequencing primer regions with the 16S region, whereas the reverse primer was simply one of 12 Illumina indices.
16S rRNA amplification of the V4-V5 hypervariable regions was conducted at the Marine Biological Laboratory in Woods Hole, Massachusetts, USA for microbial profiling using both forward and reverse primers (20 (link), 21 (link)). Due to the use of both forward and reverse primers, 96 samples were multiplexed per lane. PCR was then conducted using triplicate samples of 33 μL, and a combination of 1.0 U Platinum Taq Hi-Fidelity Polymerase (Life Technologies Carlsbad, CA, USA), 1X Hi-Fidelity buffer, 200 μM dNTP3 PurePeak DNA polymerase mix (Pierce Nucleic Acid Technologies Milwaukee, WI, USA), 1.5 mM MgSO4 and 0.2 μM of each primer. Lastly, qPCR was used to quantify each library pool before sequencing using paired-end Illumina MiSeq 100 cycle runs.
16S rRNA amplification of the V4-V5 hypervariable regions was conducted at the Marine Biological Laboratory in Woods Hole, Massachusetts, USA for microbial profiling using both forward and reverse primers (20 (link), 21 (link)). Due to the use of both forward and reverse primers, 96 samples were multiplexed per lane. PCR was then conducted using triplicate samples of 33 μL, and a combination of 1.0 U Platinum Taq Hi-Fidelity Polymerase (Life Technologies Carlsbad, CA, USA), 1X Hi-Fidelity buffer, 200 μM dNTP3 PurePeak DNA polymerase mix (Pierce Nucleic Acid Technologies Milwaukee, WI, USA), 1.5 mM MgSO4 and 0.2 μM of each primer. Lastly, qPCR was used to quantify each library pool before sequencing using paired-end Illumina MiSeq 100 cycle runs.
