Infinium hd assay ultra protocol guide

All samples were genotyped on the high-density Brassica 60K Illumina Infinium SNP BeadChip array [39 (link)], which was outsourced to TraitGenetics GmbH (Gatersleben, Germany), including DNA extraction. Samples were processed according to the manufacturer’s protocol (Infinium HD Assay Ultra Protocol Guide; Illumina [63 ]). This encompassed a series of steps (such as DNA amplification, hybridization of sample DNA onto the bead chip and imaging of the bead assay) described in detail by Gunderson, et al. [64 (link)]. Illumina GenomeStudio software (2015 v2.0.4; Illumina, Inc., San Diego, CA, USA) was used to cluster and visualize SNP array data with default clustering parameters for further analysis.

Primer sequences for the SSR markers used for genetic map construction were described by Cai et al. [33] (link) and the sequence information of all SSR markers is provided in Table S2.
The genotyping of SNPs was performed using 6K Illumina Infinium HD Assay SNP arrays of B. napus (Illumina Inc., San Diego, CA) developed by the University of Queensland. The high-quality DNA was extracted from young leaf tissues as described by Porebski et al. [60] . Each DNA sample was diluted to a final concentration of 50 ng/ µl using ddH₂O. The SNP genotyping was carried out in accordance with the Illumina protocol (Infinium HD Assay Ultra Protocol Guide, http://www.illumina.com/).
All the SNP array data were analyzed using the Illumina GenomeStudio software (Illumina Inc., San Diego, CA), which were clustered and visualized for further analysis. Each SNP was re-checked manually to determine if any error was observed during the clustering analysis. The data processing is described by Raman et al. [39] .

An antagonist protocol for controlled ovarian hyperstimulation was used for each participant. Treatment with recombinant human FSH (Gonal-f; Merck Serono, Geneva, Switzerland) was initiated on the 2nd or 3rd day of the menstrual cycle with a starting dose of 150 − 300 IU/day adjusted for age, BMI, AFC, FSH, and AMH levels. Gonadotropin-releasing hormone antagonist (Cetrotide; Merck Serono) was administered at a dose of 0.25 mg/day when the dominant follicle reached 14 mm in size or the serum E2 level reached 350 pg/mL. This treatment continued until a leading follicle reached 18 mm or two follicles reached 16 mm in size. Subsequently, 5,000 − 10,000 IU of human chorionic gonadotropin (Livzon, Zhuhai, China) was administered as a trigger and oocytes were retrieved 36 h later. ICSI and blastocyst culture were performed for all participants in accordance with IVF laboratory guidelines, and single nucleotide polymorphism microarray based PGT-A was administered to the patients with RPL as per the manufacturer’s instructions (Infinium HD Assay Ultra Protocol Guide, Illumina Inc., San Diego, CA, USA). Mosaicism calls were made when 20–80% of the biopsied cells were aneuploid.