Beadxpress platform

Genomic DNA of LARC patients was extracted from peripheral blood samples using the automated extractor BioRobot EZ1, in association with the Kit “EZ1 DNA Blood Kit 350μl” (Qiagen SPA, Milano, Italy) and stored at +4°C until the time of this study.
The selected 144 SNPs were analyzed using the Illumina BeadXpress platform, that is based on Golden Gate technology. The VeraScan software (version 2.0) was applied for fluorescence detection. GenomeStudio software 2010 (Illumina Inc.) was applied for genotype clustering, with a SNP call-threshold of 0.25 (on a scale of 0-1). Clusters were visually inspected and manually reviewed to ensure high quality data. The control dashboard was checked to evaluate the overall quality of the performed analyses and to exclude samples with low quality. Negative and positive controls were included.
Regions containing the SNPs of interest were amplified using the PCR primers designed according to Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/). Dye-terminator cycle sequencing was performed using the BigDye terminator v3.1 cycle sequencing kit (ThermoFisher Scientific). The amplicons were run on an ABI PRISM 3130xl Genetic Analyzer (ThermoFisher Scientific) and the results were analyzed with Gene Scan software (ThermoFisher Scientific). Primers and PCR conditions are available upon request.

For this study, 576 SNPs localized in 77 genes related to immune system activity and analyzable with the BeadXpress platform (Illumina, Inc., San Diego, CA, USA) were defined (Figure S1).
Genes connected with the immune system and cancer were selected with a literature analysis. The blocks of SNPs with strong linkage disequilibrium (r² ≥ 0.80) and MAF ≥ 0.05 in the Caucasian population were defined, and their genomic coordinates were analyzed with Haploregv2 software. Polymorphisms were prioritized according to their biological effect reported in the literature or predicted according to the SNPinfo web server (https://snpinfo.niehs.nih.gov/).
Polymorphisms with a high final score (≥0.7) and optimal designability (=1) according to the Illumina assay design tool were selected.

The F₅ and F₇ populations were genotyped using the 384-single nucleotide polymorphism (SNP) BeadXpress cultivar optimised genotyping panel, as described by Moragues et al. [14 (link)] and the genotyping-by-sequencing (GBS) method of Elshire et al. [15 ]. For the 384-SNP assay, leaf material from five 3-week old seedlings per line was pooled for the F₅ population. As the population was developed to the F₅ generation, the decision to pool the material was undertaken with the aim of accounting for any residual heterozygosity in individual seedlings. Genomic DNA was extracted using a Qiagen DNeasy 96 Plant Kit and genotyping using the 384-SNP assay was undertaken using the Illumina BeadXpress platform at the James Hutton Institute, Dundee, Scotland. SNP calls were analysed using Illumina BeadStudio software. From the SNP data the percentage heterozygosity in the population was as expected (6%). Due to the assumed lower heterozygosity in the F₇ population, as this is a more genetically advanced population, leaf material from a single 3-week old seedling per line was sampled. After genotyping, the level of heterozygosity in the population was calculated to be as expected for an F₇ population (<2%).

A total of 384 SNPs, previously identified for P. vulgaris (Müller et al., 2015 ) polymorphic
between BAT 93 (Mesoamerican) and JALO EEP558 (Andean) lines, was genotyped by Vera
Code^® technology with Bead X press platform (Illumina) and selected to
compose the oligopool assay (OPA).
Three oligonucleotides were used for each of the variations of the same SNP and the
third specific-locus binding to the 3’ region of the DNA fragment containing the
target SNP, generating a unique allele-specific fragment. Subsequently, this fragment
was amplified using Taq DNA polymerase enzyme Titanium (Clontech®)
and complementary primers labeled with Cy3 and Cy5
fluorophores.
Genotyping was realized by Genome Studio software version 1.8.4 (Illumina, EUA) using
Call Rate values ranging from 0.80 to 0.90 and GenTrain ≥ 0.26 for SNP grouping.
Automated analyses were performed to cluster the SNP alleles of each line, based on
the signal intensity for Cy3 and Cy5 fluorophores,
resulting in three genotype classes, AA, BB, and AB. Groups were adjusted
individually and manually by determining the best clusters based on the parental
profile.

For details on the genetic analyses, please refer to Cronjé, Nienaber-Rousseau (39 (link)). In short, genomic DNA was isolated from buffy coat using the FlexiGene™ kits (QIAGEN Inc., Valencia, CA, USA). SNPs selected from the literature [FGB-rs7439150, rs1800789 (1420G/A), rs1800791 (−854G/A), rs1800790 (−455G/A), rs1800788 (249C/T), rs1800787 (−148C/T), rs4220, rs4463047, FGA-rs6050, rs2070011 (2224G/A), and FGG rs2066865 and rs1049636 (9340T/C) as well as FXIII His95Arg A/G (rs6003) and Val34Leu, C/A (rs5985)], and two novel FGB SNPs in the promoter region (rs2227385 and rs2227388) identified via sequencing a subpopulation were genotyped. The Thermo Fischer Scientific® Taqman based assay, Illumina® VeraCode GoldenGate assay technology using a BeadXpress® platform and competitive allele-specific polymerase chain reactions (KASP) were used for genotyping.

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).