Polymerase Chain Reaction (PCR) Amplification and 16S rRNA Gene Sequencing

Fecal microbial composition was determined by Illumina MiSeq paired-end sequencing of the V3-V4 hypervariable region of the 16S rRNA gene (MiSeq Bentchtop Sequencer, Illumina Inc., San Diego, USA). Amplification of bacterial DNA was performed by PCR using modified 341F and 806R primers with a six-nucleotide barcode on the 806R primer for multiplexing (18, 19). Both primers contain an Illumina MiSeq adapter sequence, which is necessary for flow cell binding in the MiSeq machine. Primer sequences can be found in Supplementary Table S1 . A detailed description of the PCR amplification procedure, DNA clean-up and MiSeq library preparation using a 2x300 cartridge can be found in the Supplementary Methods . Demultiplexing and clustering of sequencing reads was performed using Quantitative Insights In Microbial Ecology (QIIME) with UCLUST v.1.2.22q at 97% similarity (20, 21). Taxonomic profiling was performed using Paired-eND Assembler for DNA sequences (PANDAseq) (22). All sequences with quality scores <0.9 were discarded by PANDAseq to increase sequence read-out quality, and read numbers per sample were rarefied to 25,000 read/sample. QIIME was used to assign bacterial taxonomy down to family and genus level, and ARB was used to identify sequences further down to species level (23).