title: 16S rRNA gene amplicon: feces
design: Library preparation was done using a semi-automated workflow using a Biomek4000 pipetting robot (Beckman Coulter) to enhance reproducibility and efficacy. It is preceded by a DNA extraction protocol optimized for fecal samples.  After DNA extraction, the samples are normalized to 12 ng/L according to Nanodrop measurements. 2L is used as template for the first PCR step, which is specific for amplifying the V3-V4 regions of 16S rRNA genes. A blank extraction control and a PCR negative control were included. 2L of PCR1-products are used as template for the second PCR step. The primers of the second PCR bind regions in the first primer set, and they contain the sample-specific indices that enable multiplexing and the Illumina adapters for binding to the flow cell. Both PCRs are done in duplicates to reduce primer selection bias. The duplicates are pooled per sample before a gel electrophoresis is run to check for amplification success. The pooled PCR products are cleaned using AMPureXP beads. DNA concentrations in the resulting, cleaned sequencing libraries are determined by a fluorometric method before samples are normalized to 2nM and equal volumes are pooled for sequencing.