Infinium hd assay ultra

Genomic DNA was extracted from whole venous blood using either the phenol-chloroform extraction method or the automated DNA extraction platform TECAN Freedom EVO (TECAN Group Ltd., Männedorf, Switzerland) based on the paramagnetic particle method. DNA concentration and quality were measured by a NanoDropR ND-1000 spectrophotometer (NanoDrop Technologies Inc., USA).
SNP genotyping of 254 samples was performed with Illumina HumanOmniExpress-12 v1.1 arrays at the Department of Human and Medical Genetics, Faculty of Medicine, Vilnius University, Lithuania, while the other 32 samples underwent this procedure with a HumanOmniExpress-12 v1.0 (Illumina, San Diego, CA, USA) at the University of Tartu, Estonia using the standard Illumina Infinium® HD Assay Ultra protocol recommended by the manufacturer (Catalog # WG-901–4005).
Genotyping data quality control was performed according to the standard recommendations by the manufacturer. Individuals with a call rate <98 % and a standard deviation (SD) of the log R ratio (LRR) of >0.3 were excluded from further analysis.

A total of 197 animals from the BC1_LD (160 backcrossed individuals and their respective parents) were genotyped with the Porcine SNP60 Beadchip (Illumina), following the Infinium HD Assay Ultra protocol (Illumina)56 (link). Raw data were visualized with GenomeStudio software (Illumina) and trimmed for high genotyping quality (call rate >0.99). Markers with minor allele frequency (MAF) >5% and animals with missing genotypes <5% were retained. After the quality control filter, a subset of 40,586 SNPs and 197 animals remained.
Furthermore, the BC1_LD animals were genotyped with two SNPs within the ACSM5 (rs331702081) and IGF2 (IGF2-intron3-G3072A) genes using Taqman OpenArray^TM genotyping plates custom-designed in a QuantStudio^TM 12K flex Real-Time PCR System (ThermoFisher Scientific) (Supplementary Table S7) and the pyrosequencing protocol previously described27 (link), respectively.

A total of 364 pigs, including their F0, F1 and F2 founder generations (72 animals), were genotyped with the Porcine SNP60K BeadChip [75] (link) following the Infinium HD Assay Ultra protocol (Illumina Inc.; San Diego, CA, USA) and the genotypes were visualized with the GenomeStudio software (Illumina Inc.; San Diego, CA, USA). The quality control of the 62,163 SNPs was performed by using Plink [76] (link) software removing markers with a minor allele frequency (MAF) <5% and animals with missing genotypes>5%. The SNP mapping and annotation was performed by using the pig assembly 10.2 [ftp://ftp.ncbi.hlm.nih.gov/genomes/Sus_scrofa/GEF/]. We also excluded markers which did not map in the Sscrofa10.2 version assembly. Pedstats program [77] (link) was used to check Mendelian inheritance errors.

DNA was isolated from blood using a standard phenol/chloroform protocol and genotyped with the Illumina Porcine SNP60 BeadChip [25 (link)] and the Infinium HD Assay Ultra protocol (Illumina Inc.). Genotypes of 62,163 SNPs were called with the GenomeStudio software (Illumina). In addition, DNA from 17 Iberian pigs representing the main breeding nuclei of this breed were analyzed to identify SNPs of good quality that were monomorphic or had very low minor allele frequency (MAF) in the Torbiscal line. Quality control of genotypes was performed according to the following criteria: call rate for the individual >0.96; SNPs with a call rate >0.99; GenTrain score (measure of the reliability of the SNP detection based on the distribution of genotypic classes) >0.70; AB R mean (mean of the normalized intensity of the heterozygote cluster) >0.35; and MAF >0.05. SNPs located on sex chromosomes, those not mapped in the Sscrofa10.2 assembly (http://gbi.agrsci.dk/pig/sscrofa10_2_annotation/), or those with inconsistent inheritance from dam to daughter were also removed. Based on these criteria 35,023 SNPs were retained and used for further analyses.

The total of the potato panel (183 genotypes) was genotyped using the new Illumina Infinium 12 K V2 Potato Array [24 (link), 25 (link)]. The V2 array contains 12,808 SNPs, the set of markers from the previous Infinium 8303 Potato Array and additional SNPs selected for genome coverage and on candidate genes and regions with resistant genes. The arrays were read using the Illumina iScan Reader with Infinium® HD Assay Ultra (Illumina Inc., San Diego, CA). Genome Studio 2011.1 software (Illumina, San Diego, CA) was used to assess initial sample quality. Tetraploid (5-cluster) genotyping was based on theta value thresholds, using a custom script from the Solanaceae Coordinated Agricultural Project-SolCAP [14 ]. Following 5-cluster calling, a filtering process was carried out to identify 4859 high-quality markers. These markers were used for further molecular analysis.

The 79 accessions were genotyped with 12 nuclear SSR (nSSR) markers. These markers are listed in Table S2 and encompass the nine used for grapevine identification internationally (VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62, VrZAG79)¹⁷ , plus ISV2 (VMC6e1), ISV4 (VMC6g1) and VMCNG4b9^{18 (link)}. The nSSR profiles were obtained using fluorescent primers and an ABI3130xl genetic analyser (Applied Biosystems, Foster City, CA). SSR allele calling was performed with GeneMapper software version 5.0, with a home-made bin set produced with reference varieties. Identifications were made by comparing the obtained genetic profiles with the CREA Viticulture and Enology molecular database, literature information and the Vitis International Variety Catalogue (VIVC, http://www.vivc.de).
Chlorotypes were determined with eight chloroplast SSR markers out of the nine proposed^{19 (link)}. Two multiplex PCR were organized using fluorescent primers and SSR allele calling was as described for nSSRs.
All accessions were genotyped using the Infinium® II Vitis18k SNP array, which includes 18,071 SNPs, (GrapeReSeq Consortium, Illumina) following the Infinium® HD Assay Ultra protocol (Illumina Inc., San Diego, CA) and scanned using an Illumina HiScan.