Fecal Microbiome Profiling via 16S rRNA Sequencing

DNA was extracted from fecal pellets using the UltraClean Fecal DNA Kit (MO BIO Laboratories). Amplicons were generated using oligonucleotide primers that target approximately 300 bp of the V4 variable region of the 16S rRNA gene (primers 515F and 806R) ^{40 (link)} and also were barcoded and pooled to construct the sequencing library, followed by sequencing with an Illumina MiSeq instrument to generate paired-end 150×150 reads. The software package QIIME 1.7.0 was used to analyze, display, and generate figures of microbiome data using a previously defined method^{41 (link)}. Bacterial DNA of Enterobacteriaceae and Bacteriodetes from fecal content was quantified using six-point standard curves constructed with reference bacteria specific for each bacterial group measured by qPCR in the 7500 Fast Sequence Detection System (Applied Biosystems). All the reactions were set up in Fast SYBR Green Master Mix (Applied Biosystems) at 20 µL total volume. Copy number of Enterobacteriaceae and Bacteriodetes was normalized to copy number of universal bacteria. The initialization step was 95°C for 10 minutes, the amplification step had 40 cycles of 95°C for 10 seconds followed by optimal annealing temperature for 45 seconds. The primers and reaction conditions are listed in Table S1.