16S rRNA: Prader-Willi Syndrome (PWS)
DNA samples were frozen at -20C until use. The 16S rRNA V3-V4 library was constructed by two rounds of PCR with the following primers: 341F:5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGAGGCAGCAGCCTACGGGNBGCASCAG3 and 805R:5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGACTACNVGGGTATCTAATCC3 via reaction procedure (95C for 2min, followed by 25 cycles at 95C for 30s, 55C for 30s, and 72C for 30s, and a final extension at 72C for 5min). PCR products were purified with 1x KAPA AMPure beads (KAPA, Cat#KK8002). Then, products were put through a second PCR reaction procedure (95C for 2min, followed by 8 cycles at 95C for 30s, 55C for 30s, and 72C for 30s, and a final extension at 72C for 5min). PCR products were purified with 1x KAPA AMPure beads and analyzed using a Bioanalyzer DNA kit, followed by quantification with real-time PCR. DNA libraries were pooled and sequenced on Illumina MiSeq (Illumina; CA) using a 2x250 bp paired-end protocol with overlapping reads