Iplex genotyping platform

DNA from adjacent non‐tumoral FFPE tissue from 101 cases was extracted using AllPrep DNA/RNA FFPE kit® (Qiagen, Hilden, Germany) following the manufacturer's recommendations. Samples were sent to the University of Minnesota Genomics Center for genotyping of 106 autosomal Ancestry‐Informative Markers (AIMs),²³ in a Sequenom iPLEX® Genotyping Platform. Single nucleotide polymorphisms (SNPs) with call rate lower than 90% were removed, leaving 101 for ancestry estimation; similarly, 25 samples with a call rate lower than 85% were excluded, remaining 76 cases. The concordance score for genotyping was 97.4% between 22 duplicated samples. Additionally, all AIMs were in Hardy–Weinberg equilibrium. These analyses were done in PLINK® v1.90b4.1 64‐bit. Finally, proportions of European, African, and Indigenous American genetic ancestry were estimated for each case with the ADMIXTURE® software V1.3.0 under an admixture model. To perform a supervised analysis, three parental reference populations were included: European (42 individuals from Coriell's North American Caucasian panel), African (37 non‐admixed Africans living in United Kingdom and South Carolina—USA), and Indigenous Central American populations (15 Mayan and 15 Nahuas).²³

DNA samples were sent to the University of Minnesota Genomics Center for the genotyping of 106 autosomal Ancestry-Informative Markers (AIMs) for the estimation of individual genetic ancestry (37 (link)), in a Sequenom iPLEX^® Genotyping Platform. Setting a call rate of 90% for the Single Nucleotide Polymorphisms (SNPs), 101 AIMS were suitable for ancestry estimation, and samples with a call rate under 85% excluded 31 samples from the analysis. All AIMs were in Hardy-Weinberg equilibrium. Estimation of proportions of genetic ancestry was performed as previously described (38 (link)). For each case, the proportions of European, African, and Indigenous American ancestry were estimated under an admixture model with the ADMIXTURE^® software V1.3.0. A supervised analysis was performed with three parental reference populations, which were kindly provided by Dr. Laura Fejerman: European (42 individuals from Coriell’s North American Caucasian panel), African (37 non-admixed Africans living in the United Kingdom and South Carolina - USA), and Indigenous Central American populations (15 Mayan and 15 Nahuas) (37 (link)).

Leukocyte DNA was extracted from 5 mL venous blood of patients using the EZNA blood Midi Kit (Omega Bio‐Tek, Norcross, GA, USA) according to the manufacturer's recommendation. The candidate functional SNPs in CPFL genes were selected using a set of web‐based SNP selection tools (http://snpinfo.niehs.nih.gov/snpfunc.htm) as previously described.16 Finally, nine potential functional SNPs, including three in CLOCK gene, two in BAML1 gene, and four in NPAS2 gene, were selected for genotyping with Sequenom iPLEX genotyping platform (Sequenom Inc., San Diego, CA, USA) according to the manufacturer's protocol. Strictly quality controls were performed in each assay during genotyping. SNPs with a call rate >98% were included for further analysis.