Hydrocycler 16

As described in detail in our previous study, whole genome amplified (WGA) DNA from peripheral blood leukocytes was used [29 (link)]. All WGA samples were genotyped for two common SNPs: less than 0.1% of the genotypes could not be determined or they did not agree with the corresponding genomic DNA sample, confirming the accuracy of WGA. KASP (LGC Genomics) or TaqMan (Applied Biosystems) allelic discrimination methods were used to genotype the selected SNPs. DNA amplification was performed according to the LGC Genomics’ and TaqMan´s PCR conditions. Case and control samples were amplified simultaneously in 384-well format using Hydrocycler 16 (LGC Genomics). Endpoint genotype detection was carried out on the ViiA 7 Real-Time PCR System (Applied Biosystems). The sample set contained 142 duplicated samples as quality controls. The genotype correlation between the duplicate samples was > 99%.

TaqMan SNP Genotyping Assays (Applied Biosystems) or KASP genotyping assays (LGC Genomics) were used for the analysis of the SNPs. Case and control samples were amplified simultaneously in 384 well format (Hydrocycler 16 (LGC Genomics), using 3 ng whole genome amplified DNA from blood). Endpoint genotype detection was carried out on the ViiA 7 Real-Time PCR System (Applied Biosystems). Call rates for 40 out of 41 SNPs were 94–99%. Internal quality controls showed a concordance rate of ≥ 99%. Samples with < 50% call rate over all assays were excluded from the study.

Genotyping was performed using the One Step PACE-RT™ (PCR Allele Competitive Extension) kit (3CR Bioscience) scaled for 1,536 plate format, the approach is described in Fig 2.
Each One Step PACE-RT™ SNP genotyping reaction was performed using 2.5 ng RNA, 0.005 μL One Step RT-enzyme, 0.5 μL One Step PACE-RT genotyping master mix (3CR Bioscience) and 0.018 μL assay mix (12 μM of each forward primer, 30 μM reverse primer) in a total volume of 1 μL. The combined reverse transcription and DNA amplification reaction was performed using a Hydrocycler-16 (LGC Genomics, UK) under the following conditions: 50°C for 10 minutes; 94°C for 15 minutes; 10 cycles of 94°C for 20s, 65–57°C for 60s (dropping 0.8°C per cycle); 35–40 cycles 94°C for 20s, 57°C for 60s. Fluorescence detection was performed at room temperature using a BMG Pherastar® scanner fitted with Fl 485/520, Fl 520/560 and Fl 570/610 optic modules. Genotype calling was performed using the Kraken software package version 11.5 (LGC Genomics). Fluorescent intensity was normalised for pipetting volume using the ROX standard contained within the PACE-RT master mix.