metagenome+stool: infant female
The V3 and V4 regions of the 16S rRNA gene were amplified by PCR. The first PCR amplified the V3-to-V4 region of the 16S rRNA gene using the 341F and 806R primers: 5-CCTACGGGNGGCWGCAG-3 (forward primer) and 5- GGACTACHVGGGTATCTAAT -3 (reverse primer), where the barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate 50 L mixture containing 5 L of 10  KOD Buffer, 5 L of 2.5 mM dNTPs, 1.5 L of each primer (5 M), 1 L of KOD Polymerase, and 100 ng of template DNA. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturers instructions and quantified using QuantiFluor-ST (Promega, U.S.). Purified amplicons were pooled in equimolar and paired-end sequenced (2  250) on an Illumina platform according to the standard protocols.