Taqman rnasep copy number reference assay

Genotyping of the 298bp HCG26 AluY insertion was performed by amplifying the surrounding region using primers flanking the insertion site (S1 Table). PCR was performed using 1X PCR buffer (Quanta Biosciences Inc.), 1.0mM MgCl₂, 200uM dNTPs, 500nM forward and reverse primers, 1U AccuStart II Taq polymerase (Quanta), and 100ng of DNA. Products were amplified on a 2720 thermal cycler (Applied Biosystems) at 94°C for 1 minute, 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 90 seconds. Alleles containing the insertion yielded a 1697bp product, while alleles without the insertion yielded a 1399bp product, which were resolved on a 1.5% agarose gel. Genotyping of the IL22 CNV was performed using an inventoried Taqman Copy Number Assay (Hs00146600_cn), Taqman RNAseP Copy Number Reference Assay, and 1X Taqman Genotyping Master Mix (Life Technologies) following the manufacturer’s instructions and cycling conditions. Assays were performed in quadruplicate on a 7900HT system and analyzed using CopyCaller v2.1 software (Life Technologies).

Cases and control samples were drawn from the United Kingdom Depression Case-Control (DeCC) study [29 (link)] and the Genome-Based Therapeutic Drugs for Depression (GENDEP) study [30 (link)]. mtDNA copy number was estimated from DNA extracted from blood samples by qPCR, using a TaqMan Universal PCR MasterMix on an ABI StepOnePlus Real-Time PCR System (Life Technologies). The pre-designed TaqMan assay Hs02596867_s1 was used to amplify a fragment of the MT-CYB gene on the mitochondrial chromosome in duplex with the TaqMan RNaseP Copy Number Reference Assay (Life Technologies, part number 4403326) as an internal control.