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Supplementary Table-1(a): Reagent Volumes for TAQMAN Genotyping assays, per
plate using TAQMAN buffer.
Reagent Volume (ul)
TAQMAN Mastermix (Life
technologies)
410
Nuclease Free Water (Sigma) 389.5 / 399.8
40x/80x SNP Specific Assay (Life
Technologies)
20.5 / 10.3
The volume of SNP specific assay (which contains primers and probes) depends on
whether it is supplied as 40 or 80x.
Supplementary Table-1(b): Reagent Volumes for KASPar genotyping assays per plate.
Reagent Volume (ul) for 1.8ul per
well
Volume (ul) for 3.6ul per
well
KASP Master mix 422.4 844.8
Nuclease free Water
(Sigma)
422.4 844.8
KASP Assay Mix 11.7 23.4
Supplementary Table-2(a):PCR conditions for TAQMAN Genotyping assays
Temperature Time
500C 2 mins
950C 10 mins
950C 15 sec x 40 cycles
600C 1 min x 40 cycles
Supplementary Table-2(b) PCR conditions for KASPar genotyping assays
Temperature
Decreasing 0.80C per
cycle
Time
940C 15mins
940C 20 sec x 10 cycles
650C-570C 60 sec x 10 cycles
940C 20 sec x 26 cycles
570C 60 sec x 26 cycles
Supplementary Table-2(c) PCR conditions when further cycling is required for KASPar
Genotyping Assays
Temperature Time
940C 20sec x 3 cycles
570C 60sec x 3 cycles
Supplementary Table-3(a): Sequencing pre-amplification reaction
Reagent Volume (ul)
Multiplex PCR Mastermix (Qiagen) 10
Nuclease Free Water (Sigma) 6
Forward Sequencing primer 1
Reverse sequencing primer 1
Template DNA (5ng/ul) 2
Supplementary Table-3 (b): PCR cycling conditions for sequencing pre-amplification
reactions
Temperature Time
950C 15 mins
940C 1 min x 35 cycles
65.50C 1 min x 35 cycles
720C 1 min x 35 cycles
720C 5 mins
Supplementary Table-3(c) : Sequencing Primers
Primer Sequence
MIA3_F 5’-ATCCAATCACCTTCCACCAG-3’
MIA3_R 5’-CCCAAATGTATCAGCAGCAAA-3’
CDKN2A(9p21)_F 5’-GTTTCTGCACATGGTGATGG-3’
CDKN2A(9p21)_R 5’-CATTCCCCAACATTTGTCCT-3’
APOA5_F 5’-GCAGGGTGAAGATGAGATGG-3’
APOA5_R 5’-TAGACGGAGTGGGTGTGTCA-3’
LPArs379_F 5’-GAAGGGGCTGGACCATATTT-3’
LPArs379_R 5’-AAGACCACAGGTGAGCGAGT-3’
LPLrs328_F 5’-CTTCCACAGGGTGATCTTCTG-3’
LPLrs328_R 5’-CATGAAGCTGCCTCCCTTTAG-3’
PCSK9_F 5’GACTACGAGGAGCTGGTGCT-3’
PCSK9_R 5’-CCTGCACTCCACTTCCTCTC-3’
MRAS_F 5’-TCTTGCTGCGTTTTCACATC-3’
MRAS_R 5’-TTGACTCCAAGGGAAGATGG-3’
APOB_F 5’-GCCCAGAATCTGTACCAGGA-3’
APOB_R 5’-TGGAATCTGGGGAAGTTCAG-3’
CXCL12_F 5’-GTCCAGATGAGGCCATCAAG-3’
CXCL12_R 5’-TGCCAAGAAAATGACACAGC-3’
LPArs104_F 5’-GCATAGCCAGACATGGGTTT-3’
LPArs104_R 5’-TGCCATGTTTGTCTTGGGTA-3’
APOE158_F 5’-CTGCGTAAGCGGCTCCTC-3’
APOE158_R 5’-CTGCCCATCTCCTCCATC-3’
APOE112_F 5’-GCCTACAAATCGGAACTGGA-3’
APOE112_R 5’CAGCTCCTCGGTGCTCTG-3’
F= forward; R=reverse
Supplementary Table-4: Selected CAD SNPs (Beaney et al.,2015)
Gene SNP SNP location Risk Allele Odds Ratio ReferenceMIA3 rs17465637 Intergenic C 1.14 Samani et al. CXCL12 rs1746048 Intergenic C 1.17 Samani et al. APOB rs1042031 E4181K A 1.73 Casas et al. LPA rs10455872 Intergenic G 1.70 Clarke et al. LPL rs328 S447X C 1.25 Casas et al. 9p21 rs10757274 Intergenic G 1.29 Samani, ,et al.PCSK9 rs11591147 R46L G 1.43 Benn et al. APOA5 rs662799 Promoter G 1.19 Sarwar et al. MRAS rs9818870 Intergenic T 1.15 Erdmann et al. LPA rs3798220 I1891M C 1.92 Clarke et al. APOE rs429358 C112R C 1.06 Bennet et al.APOE rs7412 C158R T 0.80 Bennet et al.SORT1 rs646776 Intergenic A 1.19 Samani et al.
Supplementary Table-5:- Multivariate analyses showing odds ratio of cytokines and
cytokine ratios for the prediction of premature atherosclerosis.
B S.E Exp(B) (95%CI) Sig.
IL-6(ng/dl) 1.867 0.648 6.470 (1.819-23.0)** 0.004
IL-18(pg/ml) 0.010 0.004 1.010 (1.003-1.018)** 0.006
IL-10 (pg/ml) 0.316 0.287 0.729 (0.415-1.281) 0.272
TNF-alpha(pg/
ml)
0.606 0.182 1.832 (1.282-2.618)** 0.001
TNF-alpha/IL-10 0.495 0.135 1.641 (1.261-2.137)** 0.000
IL-18/IL-10 ratio 0.003 0.003 0.997 (0.992-1.002) 0.270
IL-18 = Interleukin-18; IL-10 = Interleukin-10; TNF-alpha = Tumor Necrosis Factor-alpha; SNP = Single Nucleotide Polymorphism.
** p<0.01
* p<0.01
Supplementary Fig 1(a) Electropherogram showing the sequencing results for LPA
rs3798220. The genotypes for the samples are TT and CT respectively highlighted in
blue.
Sequencing primers used were:-
Forward: 5’-GAAGGGGCTGGACCATATTT-3’
Reverse: 5’-AAGACCACAGGTGAGCGAGT-3’
Supplementary Fig 1(b) Electropherogram showing the sequencing results for LPA
rs10455872. The genotype for both the samples is TT.
Sequencing primers used were:-
Forward: 5’-GCATAGCCAGACATGGGTTT-3’
Reverse: 5’-TGCCATGTTTGTCTTGGGTA-3’
Supplementary Fig 1(c) Electropherogram showing the sequencing results for PCSK9
rs11591147. The genotype for both the samples is GT.
Sequencing primers used were:-
Forward: 5’GACTACGAGGAGCTGGTGCT-3’
Reverse: 5’-CCTGCACTCCACTTCCTCTC-3’