Massarray

DNA was extracted from whole-blood samples with the use of Qiagen Maxipreps. Genotyping was performed with the use of a Sequenom MassArray matrix-assisted laser desorption/ionization-time-of-flight mass spectrometer that used Sequenom reagents and protocols. Single-nucleotide polymorphisms (SNPs) that failed Sequenom genotyping were genotyped individually with the use of TaqMan or KASPar allelic discrimination on an ABI 7900HT (Applied Biosystems) according to the manufacturer’s instructions. Of the 32 SNPs identified through genome-wide association study efforts to be associated with BMI (15 (link)), all but one (rs4836133 zinc finger protein 608) were present in MDCS, and 4 proxies (r² > 0.8) were used (rs6548238 THEM18; rs17782313 melanocortin 4 receptor; rs7498665 SH2B adaptor protein 1; and rs10913469 SEC16 homolog B, endoplasmic reticulum export factor). The mean genotype call rate was 97.1%, and all SNPs were in Hardy-Weinberg equilibrium with P > 0.0016 (0.05/31). More details on the index and proxy SNPs and genotype quality control are listed in Supplemental Table 1.

Northern Italian Centenarian Study; based on 226 centenarians (mean age 102.3) and 220 controls from Northern Italy. Lead SNPs discovered in NECS were analyzed by Sequenom MASSArray or Real Time PCR. Results for rs3184504 and rs3763305 failed the test of Hardy-Weinberg equilibrium, likely due to the small size of the cohort. We were provided with two-sided P values calculated under an allelic model.

Sickle status was diagnosed and quantification of Hb fractions was performed by high performance liquid chromatography (HPLC) using the β‐thalassaemia Short Program on the Variant I analyser (BioRad, Hercules, CA, USA). Full blood counts were performed using an automated cell counter (Pentra 60, Horiba ABX, Kyoto, Japan) within 7 d of the TCD study. Genomic DNA was isolated from peripheral blood leucocytes using Nucleon kits (BACCII). HP functional variants (alleles HP*1 and HP*2) were genotyped by allele‐specific polymerase chain reaction (PCR) adapted from published techniques (Koch et al, 2002; Cox et al, 2007). Individuals were genotyped for the 3·7 alpha‐thalassaemia deletion using a PCR‐based method and agarose gel visualization as per published methods (Williams et al, 2005). The 202‐ and 376‐single nucleotide polymorphisms (SNPs) (rs1050828 [G‐202A] & rs1050829 [A‐376G]), the combined inheritance of which results in the A‐ phenotype of glucose 6‐phosphate dehydrogenase (G6PD) deficiency, and HbS (rs334) were determined using multiplex Sequenom^® MassARRAY^® (Sequenom^®, Hamburg, Germany).

Initial assay design was carried out using Beacon Designer software (PREMIER Biosoft, CA). Probes were developed targeting wild type and all common IDH1 mutations (R132C, G, H, L and S) in central CS 12. The ability to detect all five mutations was demonstrated through analysis of tumor DNA with known mutations as assessed by at least two assays (Sequenom^™ MassARRAY, capillary sequencing, exome or sequencing). A validated single‐nucleotide polymorphism at an incidence of 0.0008–0.02% (rs148542200) was located in the optimal position for the reverse primer: for this reason two reverse primers were used to ensure assay compatibility for all patients. A synthetic long oligonucleotide was used as a template to demonstrate that both alleles amplify equally efficiently with this PCR primer strategy (data not shown; Sigma‐Aldrich, MO).
Assays targeting hotspots in MYOD1 (p.L122R), H3F3A (p.G34W), CTNNB1 (p.T41A, p.S45F) and GNAS1 (p.R201C, p.R201H) were also developed (Data S1).

The Sequenom MassARRAY and OncoCarta Panel v1.0 were used following the manufacturer's protocol (Sequenom, San Diego, CA; http://agenabio.com/oncocarta-panel) as previously described.19 The panel consisted of 24 multiplex assays capable of detecting 238 pathogenic variants in 19 oncogenes. This procedure was a rapid, cost‐effective method of identifying key cancer‐driving pathogenic variants across a large number of samples because it avoided complex bioinformatic analyses and assays were performed within 2 days. The amount of DNA added to the polymerase chain reaction was 20 ng per reaction. DNA was amplified using the OncoCarta PCR primer pools. Unincorporated nucleotides were inactivated by shrimp alkaline phosphatase, and a single‐base extension reaction was performed using extension primers that hybridize immediately adjacent to the mutations and a custom mixture of nucleotides. Salts were removed by the addition of a cation‐exchange resin. Multiplexed reactions were spotted onto SpectroCHIP II arrays, and DNA fragments were resolved by MALDI‐TOF on the Compact Mass Spectrometer (Sequenom). An additional customized panel was used for some of the samples as a quality control. Details regarding genes and hot‐spot pathogenic variants analyzed within the OncoCarta panel are provided within Table S1.

Tag-SNPs of IL-6 and IL-6R were selected following data release from Phase II of the International HapMap project [15 (link)]. Sample based genotypes were downloaded for all variants in genomic regions including from 5,000 bp 5-prime upstream to 5000 bp 3-prime downstream of IL-6 and IL-6R independently.
Since the study populations under investigation were from the Chinese population, downloaded genotypes were restricted to those for the Han Chinese in Beijing, China (CHB) population (http://hapmap.ncbi.nlm.nih.gov). Tag-SNPs were selected using a pairwise tagging algorithm by Haploview software (available at http://www.broadinstitute.org/haploview), with a correlation coefficient (r2) exceeding 0.8 for all downloaded SNPs with minor allele frequency (MAF) >5% [16 (link)]. Because the tag-SNP probabilities were discrete, accordingly, functional ranking of tag-SNPs with the same probability was used.
Blood samples from all participants were collected and stored at −20°C. Genomic DNA was extracted from peripheral leukocytes by using a Genomic DNA Extraction kit (QIAamp DNA Blood Mini Kit; Qiagen, Hilden, Germany). MassArray (Sequenom, USA) was used for genotyping selected tag-SNPs (Gabriel et al., 2009), and this assay was accomplished by Bio Miao Biological Technology (Beijing, China). The primers were designed using iPLEX GOLD (Sequenom, USA) [17 ].