Genomestudio

Using the Illumina TotalPrep RNA Amplification kit (Ambion), double-stranded cDNA was generated following reverse transcription from 100 ng of total RNA. In vitro transcription overnight with biotin-labeled nucleotides created amplified mRNA (cRNA), which was concentrated by vacuum centrifugation at 30°C. 750 ng cRNA per sample was then hybridized to Human HT-12 Expression BeadChips (Illumina) using the Whole- Genome Expression Direct Hybridization kit (Illumina). Finally, chips were scanned on the Illumina Beadstation 500. The chip annotation manifest was version 4, revision 1. For differential expression analysis and the generation of gene lists for functional annotation and pathway analysis, microarray data were processed in GenomeStudio (Illumina, V2011.1). Raw data were adjusted by background subtraction and rank-invariant normalization. Before calculating fold change, an offset of 20 was added to all probe set means to eliminate negative signals. The p- values for differences between mean signals were calculated in GenomeStudio by t-test and corrected for multiple hypotheses testing by the Benjamini-Hochberg method in combination with the Illumina custom false discovery rate (FDR) model. Microarray data have been uploaded to GEO (accession number GSE54179).

Data analysis for blood transcriptome from CA patients was performed using the following softwares: GenomeStudio (version 3.0, Illumina), GenPlex™ (version 3.0, ISTECH, Inc., Seoul, Korea), EXCEL (Microsoft), and GSEA (version 2.07, Broad Institute). Briefly, GenomeStudio (version 3.0) was used for the data acquisition and calculation of signal values on Illumina expression beadchip. Normalization of expression data and hierarchical clustering was performed by GenPlex™ (version 3.0). For primary data filtering, spots with a P-call (Detection call P-value < 0.1) were selected, and normalized via quantile normalization. A multitude of analyses was performed using the normalized and filtered data. Sets of differentially expressed genes (DEGs) were identified by combination analysis of Welch's t test and fold change, and the DEGs with a fold change deregulation of more than 1.3 and P-value < 0.05 were selected.

RNA was assayed at Scripps Genomic Medicine (La Jolla, CA, USA) for labeling, hybridization, and scanning using the Illumina BeadChips pipeline (Illumina, San Diego, CA, USA) per the manufacturer’s instruction. All arrays were scanned with the Illumina BeadArray Reader and read into Illumina GenomeStudio software (version 1.1.1). Raw data were exported from Illumina GenomeStudio, and data preprocessing was performed using the lumi package [46 (link)] for R (http://www.R-project.org) and Bioconductor (https://www.bioconductor.org) [47 (link)]. Raw and normalized data are part of larger sets deposited in the Gene Expression Omnibus database (GSE42133; GSE111175).

Human CytoSNP-12 Infinium HD BeadChips and GenomeStudio software (Illumina) were used for genome-wide single nucleotide polymorphism (SNP) genotyping and for data filtering and analysis, respectively. Copy number variation (CNV) analysis was performed using CNVPartition version 2.4.4 with a confidence threshold set at 50 and a minimum of 10 SNP probes per CNV region [26 (link)]. Specific PEX gene exons were sequenced using described protocols [27 (link)]. 450K Infinium Methylation BeadChips (Illumina) and GenomeStudio software were used for global DNA methylation analysis, as described [28 (link), 29 (link)]. DNA methylation levels were summarized as β-values ranging from 0 (unmethylated) to 1 (fully methylated). Scaled DNA methylation scores and .idat files are available at the NCBI GEO repository [25 ] under Series Accession Number GSE68134. Confirmatory bisulfite DNA sequencing was conducted as described [28 (link), 29 (link)].

The three software packages, GenomeStudio (Illumina software), ClusterCall (R package)^{7 (link)}, and polyBreedR (the function geno_call, R package) (https://polyploids.r-universe.dev/articles/polyBreedR/Vignette1.html), which have been developed to generate dosage genotype calls based on different models, were compared in terms of reproducibility for three independent replicates of the 16 Korean varieties (Table S5).
The average number of loci with contradicting calls within these replicates after filtering (call rate 0.90, MAF 0.05) was only 0.2%, with a maximum of 0.3%, in GenomeStudio, whereas in ClusterCall, the number of markers with discordant calls between replicates was only 0.4%, with a maximum of 0.8%. There were no significant differences between the two software programs.
In contrast, for the function geno_call of polyBreedR, which employed the normal mixture model implemented in the R package fitPoly, the average difference was 3.8%, with a maximum of 6.3%. Thus, ClusterCall was used to generate dosage genotype calls for the merged dataset from different sources of raw data, as described below (Table S5, Fig. S1).

Initial processing of data was performed using GenomeStudio to identify samples with detection rates dissimilar to the rest of the project (GenomeStudio, Illumina). Stringent quality control processing of the data was conducted using the standardised procedures from internal pipelines (available at https://github.com/snewhouse/BRC_MH_Bioinformatics). Expression data were background-corrected using module-based background correction for Beadarray.^{29 (link)} Probes were then filtered by selecting probes with expression levels >2 s.d. greater than the mean intensity of the negative control (background) beads. Reported gender was compared to XIST gene expression (female specific) and Y chromosome gene expression (male specific). Each probe was then transformed and normalised using log2 transformation and robust-spline normalisation.^{30 (link), 31 (link)} Sample relationships within the co-expression network were assessed and outliers were removed.^{32 (link)} Following sample outlier removal, 95 samples at pre-treatment, 99 samples at post treatment and 97 samples at follow-up remained. Probes detected in <80% of the sample were removed. Following quality control procedures, 4381 probes with high-quality data were available for analysis. All quality control was performed in R³³ making use of the lumi package.^{31 (link)}