Attention Deficit Hyperactivity Disorder and the Gut Microbiome

This data has been publicly released and includes a set of de-identified gut microbiome samples from 61 undergraduate students. Each student was assessed using the ADHD Self-Report Scale (ASRS), which has two subscales (inattention and hyperactivity). Those who scored 17 or above on either or both subscales were considered likely to have ADHD (Rosler et al, 2010). Sample names include group (Control or ADHD), gender, ASRS score (format: inattention/hyperactivity) and overall diagnosis (Control, or ADHD inattentive/hyperactive/both). Sample numbers are pseudorandom, and have no correspondence to any master key. Participants in this study were given an Omnigene-Gut OMR-200 stool sample collection kit for sample collection, which they returned on average within three days (SD=2.48). DNA sequencing was then performed at Genewiz, Inc. (South Plainfield, NJ) where DNA was extracted from a 200-microliter vortexed fecal sample solution from the Omnigene Gut Collection Tube using the Zymo BIOMICS DNA Mini-Kit. Sequencing Details (copied directly from the Zymo Research Metagenomic Services Report): For each sample, 200 microliters were used for DNA extraction, which was performed with in-house format (low bioburden) of ZR Fecal DNA Miniprep (Zymo Research, CA, US). The DNA extraction was performed in a clean room to minimize contamination by following the manufacturer's protocol. Two process controls (negative controls) were run simultaneously by replacing 200 microliters of customer samples with 200 microliters of pure water to assess the bioburden in the DNA extraction process. These two controls were profiled together with the customers' samples in the subsequent process. Bacterial 16S ribosmal RNA gene targeting sequencing was performed. The general bacterial 16S primers used were 313f (CCTACGGGNGGCWGCAG) and 806r (GACTACHVGGGTATCTAATCC), which amplified the v3-4 region of the 16S rRNA gene. The sequencing library was prepared by following a published protocol (Kozich et al, 2013) with some modifications to prevent PCR chimera formation: the number of PCR cycles is carefully controlled for each sample, so that the reaction can generate enough product and does not overrun. The amplicon libraries were cleaned up with Zymo's Select-a-Size DNA Clean and Concentrator (>200 fragments were kept), quantified with TapeStation, normalized and pooled together. The final library was quantified with qPCR and sequenced Illumina MiSeq with v2 reagent kit (500 cycles). The sequencing was performed with 10% PhiX mix and in paired-end mode. Raw sequence reads were trimmed with Trimmomatic-0.33 (Bolger et al, 2014). The two paired-end reads in each pair were assembled to construct a complete amplicon sequence with SeqPrep (https://github.com/jstjohn/SeqPrep). Chimeric amplicon sequences were identified and removed with Usearch (v. 6.1) (Edgar et al, 2011) in de novo mode. Amplicon sequences smaller than 302 bp were removed. Post-processing using differential analysis (Jackson et al, 1991) revealed three samples as outliers, fairly consistent with the "three-sigma rule" (Ruan, 2005) in the normal distribution; we recommend not including these in any analyses.