S10_P3: Female, 7  months, megacolon, distal of the colon
The V4 region of bacterial 16S rRNA was amplified from each total DNA sample using bacteria/archaeal primers: 515F and 806R as previously described by Caporaso et al., 2012. 25 ?l PCR reactions were performed using the TaKaRa ExTaq enzyme mixture (Takara Bio Inc. Shiga, Japan) and 200 ng of fecal DNA under the following conditions: step 1: 94°C for 3 min, step 2: 94°C for 45 s, step 3: 50°C for 1 min, step 4: 72°C for 1min, step 5: go to 2, 25 times, step 6: 72°C for 10 min, step 7: 4°C forever. Products were stored at 4°C. Equal quantities of each sample were pooled, and the PCR products were gel-purified and sequenced by MiSeq (Illumina) according to the manufacturers protocol. Reads were demultiplexed using Illumina software and separate fastq files were generated for each specimen.