Supplementary Document 1

DNA collection and Genotyping

Genomic DNA was extracted from buccal mucosa cells that were collected and extracted according to the protocol previously described (Freeman et al, 2003). In brief, individuals received cytology brushes (Cytobrush plus C0012, Durbin PLC) and a 15-mL plastic tube containing 2.0 ml of collection buffer by mail. They were instructed to give the sample in the morning before eating or drinking by scraping the inside of their cheeks on both sides with the brush then return the brush in the tube by mail. After extraction DNA concentration was measured with spectrophotometer (NanoDrop Technologies, USA), and samples were normalised to 10 ng/ul.

Determination of the 5HTTLPR genotype was performed as previously described, with some modifications. Polymerase chain reactions (PCR) used a forward primer (5’- GCCAGCACCTAACCCCTAAT -3’) labelled with 6-FAM (6-carboxyfluorescein) and a reverse primer (5’- GTAGGGTGCAAGGAGAATGC -3’), to give a product size of 366 bp. Reactions were performed in a Thermo-Fast® 384 PCR plate (ABgene Ltd, UK) on a PTC-220 Dyad TM thermocycler (Bio-Rad, Hercules, CA). PCR amplification was performed in a final 10 ul volume containing 0.25 uM of each primer (Metabion, GmbH, Germany), 1x NH4 buffer, 1.5 mM MgCl, 0.1 mM of each d NTP, 0.2 units of Taq DNA polymerase (all from Bioline, UK) and 20 ng DNA. Cycling conditions were as follows: initial 15 min denaturation at 95°C, then 34 cycles of denaturation at 94°C for 20 s, annealing at 64°C for 30 s and extension at 72°C for 30 s; and a final extension for 10 min at 72°C. Polymerase chain reaction products were analyzed using an ABI3100 Genetic Analyzer and GeneScan analysis software (Applied Biosystems, Nieuwerkerk aan den IJssel, the Netherlands).

The SNPs were genotyped using the Sequenom ® MassARRAY technology (Sequenom ®, San Diego). The IplexTM assay was followed according to manufacturers instructions (www.sequenom.com) using 25ng of DNA. Forward, reverse and extension primers were designed using the Assay Design 3.0 software of Sequenom. The oligos were ordered in a plate format from Metabion GmbH, Germany. The primers were rehydrated to 100 µM and the extension primers to 400 µM. The list of primers and callrates can be seen at the end of this document.

PCR was performed with 3.5 mM MgCl2, 0.5 U Hot Start Taq polymerase, Hot Start Taq PCR buffer (1.25x), 100 nM forward and reverse PCR primers, 500 µM dNTPs, and nuclease-free water. PCR thermal cycling (PTC-225 tetrad, Bio-Rad, Hercules, CA) was performed at 94°C for 15 minutes, (94°C for 20 seconds, 56°C for 30 seconds, and 72°C for 60 seconds) for 45 cycles, and finally 72°C for 3 minutes. The amplicon length is designed to be around 100 bp.

Dephosphorylation of unincorporated dNTPs was achieved by adding 0.3 µl (0.3 U) Shrimp Alkaline Phosphatase (SAP) and 0.17 µl buffer and 1.53 µl water to PCR products, and incubating at 37°C for 40 minutes, followed by inactivation for 5 minutes at 85°C.

After adjusting the concentrations of extension primers to equilibrate signal-to-noise ratios, the post-PCR primer extension reaction of the iPLEX assay was performed in a final 9 µl volume extension reaction containing 0.2 µl of iPLEX termination mix, 0.2 µl iPLEX buffer, 0.8 µl primer mix (0.625 µM LM, 1.25 µM HM), 0.04 µl iPLEX enzyme, and 0.76 µl nuclease-free water. Extension (GeneAmp PCR system 9700, Applied Biosystems, Netherlands) used a 2-step 200 short cycles program for approximately 3 hours. Samples were denatured at 94°C for 5 seconds, strands annealed at 52°C for 5 seconds, and extended at 80°C for 5 seconds. A final extension was done at 72°C for 3 minutes. The iPLEX reaction products were desalted with Clean Resin before being dispensed onto a 384-well SpectroChip (Sequenom Inc.), processed and analysed in a Compact Mass Spectrometer by MassARRAY Workstation (version 3.3) software (Sequenom Inc.).

All laboratory work was performed under the ISO 9001:2000 quality management requirements. Duplicate genotyping of 15% of samples yielded a genotyping error rate of 0.016%.

References

Freeman B, Smith N, Curtis C, Huckett L, Mill J, Craig IW (2003). DNA from buccal swabs recruited by mail: evaluation of storage effects on long-term stability and suitability for multiplex polymerase chain reaction genotyping. Behav Genet 33: 67-72.

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Supplementary Document 1

marker_ID / forward primer / reverse primer / extension primer / callrate
1 / rs1843809 / ACGTTGGATGAGGCCCTGAGCTCCTACTTT / ACGTTGGATGCCAGGAGTAGGTACATTCAT / CCCTGAGCTCCTACTTTAATTAT / 95.54%
2 / rs1386493 / ACGTTGGATGGCGTGATTATCCTACAGCTC / ACGTTGGATGTGTCACAGTGAAATGGGTGG / CCTACAGCTCATGGTC / 96.13%
3 / rs6582078 / ACGTTGGATGGTATAACCTGTGTGGGAGTC / ACGTTGGATGTTTCCCCTGACTTGCAAAGC / TGTGGGAGTCTGGAAG / 96.04%
4 / rs10506645 / ACGTTGGATGAGAATGAGTCCATAGGGTCC / ACGTTGGATGCTGAGAGGTAGATGGGTAAC / caTCCCGGCTTAGAACTA / 95.46%
5 / rs1352250 / ACGTTGGATGGATAGAACTACCTTGGTAAC / ACGTTGGATGGAAGTGTGTGCAAAGCATGG / GTTCATGTGCTGAGTTG / 95.46%
6 / rs1487275 / ACGTTGGATGAGATTGGTTTGAGATGTGCC / ACGTTGGATGTGAGGACCTGAGGGAGTATC / ATAACAAAAAGTTATAGGCTAAATT / 93.52%
7 / rs1386485 / ACGTTGGATGCATGTGCTTAATAGGCTGAC / ACGTTGGATGGAGCTCGTGGAAAAAATGTG / cTGTGCTTAATAGGCTGACCCAAAC / 93.77%
8 / 5HTTLPR / GCCAGCACCTAACCCCTAAT / GTAGGGTGCAAGGAGAATGC / na / 97.39%
9 / rs6295 / ACGTTGGATGGTCAGTCTCCCAATTATTGC / ACGTTGGATGCGAGAACGGAGGTAGCTTTT / AGACCGAGTGTGTCTTC / 96.72%
10 / rs1800532 / ACGTTGGATGCATGCTCTATATGTGTTAGCC / ACGTTGGATGGTGTTACATTCCCTATGCTC / ATTAATTGACAACCTATTAGGTG / 96.38%

The average individual callrate was 96% for these markers in the included population.

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