16s rRNA gene amplicons: human fecal microbiome
For genomic DNA isolation, QIAamp Fast DNA Stool Mini Kits (Qiagen, Venlo, the Netherlands) were used. Barcoded amplicons from the V3-V4 region of 16S rRNA genes were generated using a 2-step PCR. In the first step, 1025 ng genomic DNA was used as template for the first PCR with a total volume of 50 l using the 341F (5-CCTACGGGNGGCWGCAG-3) and 785R (5-GACTACHVGGGTATCTAATCC-3) primers appended with Illumina adaptor sequences. PCR products were purified (QIAquick PCR Purification Kit), the size of the PCR products was checked on a Fragment analyzer (Advanced Analytical, Ankeny, US), and concentration quantified by fluorometric analysis (Qubit dsDNA HS Assay Kit). Purified PCR products were used for the second PCR in combination with sample-specific barcoded primers (Nextera XT index kit, Illumina, San Diego, CA, USA). Subsequently, PCR products were purified, checked on a Fragment analyzer and quantified, followed by equimolar multiplexing, and sequencing on an Illumina MiSeq with the paired-end (2x) 300 bp protocol (Illumina, Eindhoven, The Netherlands).