16S rRNA Amplicon Sequencing of Homo sapiens stool sample
Microbial DNA was extracted using PowerSoil DNA Isolation Kit (MO BIO Laboratories), with Proteinase K addition (Qiagen) for 10 minutes at 65C prior to bead-beating using TissueLyser II (Qiagen) at 30 Hz for 2 minutes. DNA was eluted in UltraClean PCR Water (MoBio Laboratories). 16S rRNA gene (V4 region) sequencing was performed using primers (515F = GTGCCAGCAGCCGCGGTAA and 806R = GGACTACCAGGGTATCTAAT) that were modified to include a linker sequence, a 12 bp index sequence and a heterogeneity spacer [32]. PCR was set up with LA PCR Kit (Takara Bio USA). Libraries were cleaned up (Agencourt AMPure XP Kit; Beckman Coulter), quantified (Quant-IT dsDNA High-Sensitivity Assay Kit; Invitrogen) and sequenced using a 250-bp paired-end sequencing protocol on the MiSeq platform (Illumina Inc.) at the NIH Intramural Sequencing Center.