The TCGA-CESC (The Cancer Genome Atlas-Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma) project's massive RNA-sequencing gene expression matrix was retrieved from the UCSC Xena project (http://xena.ucsc.edu ) [29 (link), 30 (link)]. The platform used in this assay was Illumina Hiseq Rnaseqv2 Platform and detected 308 samples with 20531 identifiers (version 2018-09-03). Also, the clinical information of these samples is obtained from corresponding TCGA-CESC curated survival (version 2018-09-03) and phenotype data (version 2019-12-06) and utilized in the study [31 (link)].
Hiseq rnaseq v2 platform
Manufactured by Illumina
The HiSeq RNAseq V2 platform is a next-generation sequencing system designed for high-throughput RNA sequencing. The platform uses Illumina's sequencing-by-synthesis technology to generate high-quality RNA sequencing data.
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9 protocols using hiseq rnaseq v2 platform
1
TCGA-CESC RNA-seq Gene Expression Analysis
2
RNA-Seq Gene Expression Data for Cancers
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RNA-Seq by Expectation Maximization (RSEM) normalized gene expression for epithelial-based cancers and normal tissue was downloaded from The Cancer Genome Atlas (TCGA) database, which was accessed through the Firebrowse database using the ‘RTCGAToolbox’ package (version 2.20.0)39 (link) in R. The following TCGA Study Abbreviations were downloaded (exact labels found in database): ACC, BLCA, BRCA, CESC, CHOL, COADREAD, ESCA, HNSC, KIRC, KIRP, KICH, LIHC, LUAD, LUSC, MESO, OV, PAAD, PRAD, STAD, THCA, THYM, UCEC. These values were measured through the Illumina HiSeq RNAseq V2 platform and were log2 transformed.
3
Papillary Renal Cell Carcinoma Transcriptomics
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173 datasets were found in GEO series (http://www.ncbi.nlm.nih.gov/gds ) by search term “papillary renal cell carcinoma” (Searched on 10th of February 2016). After manually removing datasets in which PRCC1 and PRCC2 had not been separately classified, four datasets were obtained (Table 1 ). For the GSE26574 dataset, 22 PRCC1 and 12 PRCC2 samples were analyzed by expression profiling microarray using a GPL11433 platform without gender information [47 (link)]. In GSE7023 dataset, 14 PRCC1 and 16 PRCC2 samples were analyzed by expression profiling microarray using GPL4866 platform without gender information [48 (link)]. In GSE11024 and GSE2748 datasets, gender information was available for a total of 25 PRCC1 and 21 PRCC2 samples which were analyzed by expression profiling microarray using GPL6671 and GPL570 platforms, respectively [49 (link)–50 (link)]. In addition to microarrays datasets, one large PRCC dataset was available via TCGA-KIRP and consisted 77 PRCC1 and 86 PRCC2 with gender and clinical information [51 (link)]. This dataset had been analyzed by RNA-seq using Illumina Hiseq RNASeqV2-platform (http://cancergenome.nih.gov/ ).
4
Transcriptomic Profiling of Cancers
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Data were taken from the GDAC Firebrowse TCGA portal provided by the Broad Institute. miRNA datasets used were log2 normalised mature miRNA counts for all cancer types. mRNA datasets used were normalised RSEM genes taken from data through the Illumina HiSeq RNAseq v2 platform. These expression data were then transformed by the transformation log2(x + 1), for x as the original expression value, and this was used in all further computation for all cancer types and signatures. Where not otherwise specified, signature scores are taken as the median of log2-transformed expression of all signature genes for each sample. Metabric datasets for normalised miRNA and mRNA expression were taken from the European Genome-Phenome Archive (EGA) under study accession numbers EGAD00010000434 and EGAD00010000438 . In all analyses, only miRNA and mRNA expressed at a non-zero level in at least 80% of samples were considered.
5
GNAS and MTERF1 Methylation and Expression in Gastric Cancer
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Because of the lack of GC cases and mRNA expression in our multi-stage validation, we accessed TCGA stomach cancer study data (http://www.xenabrowser.net ) for the methylation and mRNA expression levels of GNAS and MTERF1 in GC cases. A total of 11 CpG sites in the promoter region of GNAS (from cg19640589 to cg21625881) and 15 CpG sites in MTERF1 (from cg10872641 to cg22709100) were selected from the Illumina HumanMethylation 450 DNA methylation data. The mRNA expression data were retrieved from Illumina HiSeq RNA-SeqV2 platform and log2 (RPKM+1) transformed. Other relevant clinical information was obtained including age, gender, pathological stage, H.pylori infection, targeted molecular therapy and radiation therapy.
6
Integrative Analysis of TCGA Lung Adenocarcinoma
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The omics data in TCGA for lung adenocarcinoma were used in this study, including methylation, mRNA expression, and clinical information (https://portal.gdc.cancer.gov/ ). The information of DNA methylation (level three) was generated using the Illumina Human Methylation 450 platform for all samples on 1 October 2017. The gene expression data (level three) were generated from the Illumina HiSeq RNA‐seq V2 platform. The clinical data were downloaded for survival analysis at the same time. In total, 418 patients for whom both TCGA methylation array and clinical data were available and 26 adjacent normal samples from the TCGA dataset were included in this study.
7
TCGA RNA-Seq Transcriptomic Data Pipeline
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The Cancer Genome Atlas (TCGA) database, which was accessed through the Firebrowse database using the 'RTCGAToolbox' package (version 2.20.0) 39 in R. The following TCGA Study Abbreviations were downloaded (exact labels found in database):
ACC, BLCA, BRCA, CESC, CHOL, COADREAD, ESCA, HNSC, KIRC, KIRP, KICH, LIHC, LUAD, LUSC, MESO, OV, PAAD, PRAD, STAD, THCA, THYM, UCEC. These values were measured through the Illumina HiSeq RNAseq V2 platform and were log2 transformed.
ACC, BLCA, BRCA, CESC, CHOL, COADREAD, ESCA, HNSC, KIRC, KIRP, KICH, LIHC, LUAD, LUSC, MESO, OV, PAAD, PRAD, STAD, THCA, THYM, UCEC. These values were measured through the Illumina HiSeq RNAseq V2 platform and were log2 transformed.
8
Integrative Analysis of LUAD Methylome and Transcriptome
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In this study, 1095 LUAD cases in total were downloaded from TCGA data portal (https://cancergenome.nih.gov/ , level 3， normalized gene expression data [RSEM] and HumanMethylation450 data) accessed on 20171206. Of them, 636 LUAD patients had whole genomic DNA methylation data of 485 578 CpG sites, which was profiled by using Illumina Infinium HumanMethylation450 BeadChips assay. And 576 LUAD sufferers had transcriptomic data of 20 532 genes, which was analyzed using Illumina HiSeq_RNASeq V2 platform. A total of 492 of these 576 LUAD suffers had recorded clinical annotation data and involved in further Kaplan‐Meier survival analysis.
To precisely discover the differential methylation CpG sites and transcripts, we only selected the cases which had data for both LUAD tumor and adjacent non‐LUAD normal tissues. Finally, 18‐paired cases were co‐existed in 29 coupled (tumor and adjacent tissue) methylation data and 57 coupled RNA‐seq data, which was used in the followed methylation and expression analysis (Figure1 ).
To precisely discover the differential methylation CpG sites and transcripts, we only selected the cases which had data for both LUAD tumor and adjacent non‐LUAD normal tissues. Finally, 18‐paired cases were co‐existed in 29 coupled (tumor and adjacent tissue) methylation data and 57 coupled RNA‐seq data, which was used in the followed methylation and expression analysis (Figure
9
HNSCC Gene Expression Data Analysis
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Information for the HNSCC training set was obtained from TCGA on November 13, 2016 (18 (link)). Gene expression data were extracted from IlluminaHiSeq_RNASeqV2 platform and normalized by RSEM method (19 (link)). In addition, we performed quality control with a total of 20530 genes. Genes with more than half of values as zero were removed, 17711 genes remained with quantile normalization. Patients with complete follow-up information and gene expression values for tumor tissues were included in the study. Information for the HNSCC testing set was collected from GSE65858 (20 (link)) in GEO. Gene expression data were extracted from Illumina HumanHT-12 V4.0 expression beadchip and normalized using the robust spline normalization (RSN) method (21 (link)). Consecutive patients with primary and metachronous secondary HNSCC of oral cavity, larynx, oro- and hypopharynx were included, while tumor cell lines and those with low quality assays were excluded. All gene expression values were log2-transformed and standardized for comparability between the training and testing sets.
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