Iplex matrix assisted laser desorption ionization time of flight mass spectrometry

Polymorphisms to be tested in the replication cohort were genotyped by the Canadian research team using iPLEX matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom, San Diego, CA, USA). Negative controls and a 5% random sample duplicate population were used to ensure the robustness and reproducibility of the assay. All extension primers and PCR assays were designed using Spectro DESIGNER software (Sequenom, San Diego, CA, USA). Markers that could not be sequenced due to poor primer design or because they were located in duplicated regions were replaced with TagSNPs in complete linkage disequilibrium (LD; r² = 1.00).

The polymorphisms identified as prognostic markers in this study and that need to be tested in the FOLFIRI plus bevacizumab cohort were genotyped using the iPLEX matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom, San Diego, CA, USA) as describe above.

Single-nucleotide polymorphisms in the HNF1A gene and 5 kb flanking regions were identified using the CEU population of the International HapMap Project information¹. htSNPs were selected using Haploview v4.2 to tag for SNPs in high LD (r² > 0.8) (Broad Institute, Cambridge, MA, United States) (Barrett et al., 2005 (link)). Allele frequencies and LD data from the 1000 Genomes Project Phase 3 were obtained through the 1000 Genomes Browser – Ensembl², assembly GRCh37.p13, accessed on March 22, 2017. The list of htSNPs (n = 13) and their associated SNPs is provided in Supplementary Table 1. Genotyping of htSNPs was performed by Sequenom iPLEX matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom, San Diego, CA, United States) and using the Illumina BeadXpress platform (Illumina Inc., San Diego, CA, United States). Experimental design of PCR primers, extension primers and genotyping conditions were defined with the SpectroDESIGNER software (Sequenom).