Broad range dna reagents

Upon receipt at Brown University, fecal samples were stored at −80°C until all samples had been collected. Samples were then thawed, and 300 μL of fecal suspension from each sample was transferred into two plates of the ZymoBIOMICS 96 DNA Kit (Zymo Research) to extract DNA. Samples from the two groups were randomized across the two 96-well plates. Extraction was performed according to manufacturer’s protocols, and extracted DNA was measured using the Qubit 3.0 system with Broad-Range DNA reagents (Thermo Fisher Scientific).

Amplicons of the V4 hypervariable regions of the 16S rRNA gene were generated according to the Earth Microbiome Protocol^{140 (link)}. In brief, 10 μg of extracted DNA from each sample was used as template for triplicate PCR reactions utilizing individually barcoded 515F forward primers (GTGYCAGCMGCCGCGGTAA) with Illumina adapters and the 806R reverse primer (GGACTACNVGGGTWTCTAAT) with Illumina adapters. Triplicates were combined and measured using the Qubit 3.0 system with Broad-Range DNA Reagents (Thermo Fisher Scientific). Samples were pooled in equimolar concentrations and sent out for 2x250 paired-end sequencing utilizing an Illumina MiSeq system at the University of Rhode Island. We obtained a total of 3,806,054 quality-filtered sequences across all 90 samples. The average sequencing depth was 41,509 reads in the control group and 44,169 in the MDD group. Sequences can be found at the NCBI Short Read Archive under BioProject ID PRJNA591924.