title: IBD_ITS
design: Stool samples were collected from pediatric patients with IBD, and bacterial, archaeal, and fungal community structures were characterized by deep sequencing of rRNA gene segments. The results were compared to results from a previously published cohort of healthy pediatric and adult subjects.
library_construction_protocol: Using the PSP Spin Stool DNA Plus Kit (Cat #10381102, Invitek, Berlin, Germany) according to the manufacturer=E2=80=99s instructions, DNA was isolated from approximately 200 mg of stool. Isolated DNA was quantified using the Picogreen system and 50 ng of DNA was amplified in each PCR reaction. Primers were barcoded to label each sample, like has been described in Hamady et al. 2008. Primers annealing to the V1V2 region of the 16S bacterial gene were used for amplification. The development of the archaeal specific 16S rDNA primers and ITS1 fungal primers and PCR cycling conditions are described in Hoffmann et. al 2013. Amplified 16S rDNA and ITS1 fragments were purified using a 1:1 volume of Agencourt AmPure XP beads (Beckman-Colter, Brea, CA, USA). The purified products from the stool samples were pooled in equal amounts and analyzed by pyrosequencing using the Roche/454 Genome Sequencer Junior.  DNA pools were separated by amplicon type.