Multi-omics data integration colon cancer using proteogenomics approach
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1 Dept. of Medical Oncology Multi-omics data integration colon cancer using proteogenomics approach DTL Focus meeting, 29 August 2016 Thang Pham OncoProteomics Laboratory, Dept. of Medical Oncology VU University Medical Center Featuring 2 KWF projects (co-pi Connie Jimenez), in collaboration with AMC, NKI.
2 Contents CRC proteomics data integration Proteogenomics: splice variants Lots of opportunities when raw data accessible analysis pipelines reproducible version control of external resources: Uniprot, RefSeq, etc...
3 CCA O P VUmc L ONCO Proteomics Laboratory Colorectal cancer, a heterogeneous disease Medical Oncology CRC-OMICs uncovering molecular disease subtypes Int. J. Cancer 2014 Nature 2014
4 TCGA colorectal cancer proteomics 95 samples (5 replicates) 95x15 raw Orbi. Velos files peptides 7526 proteins 86 matched RNA-seq Nature medicine 2015 Nature 513, (2014) Proteogenomic characterization of human colon and rectal cancer.
5 In-house datasets Medical Oncology 2014: TCGA 95 protein profiles 5 subtypes 2013: 1100 gene expression CCS1, CCS2, CCS3 end 2014: 29 tissue profiles + 18 cell lines [OPL47] 2015: CMS1, CMS2, CMS3, CMS4 70 candidates (mostly CCS1 vs CCS3) 2016: 9 CMS2 + 9 CMS3 [OPL18] 2016: 39 single shots CMS1-4 *37 biological samples Identify discriminatory CMS subtype-associated proteins & generate protein classifier 2016: ~120 single shots CMS1-4
6 Medical Oncology Datasets & pipelines OPL 29 tissues + 18 cell lines 217 (=29x5 + 18x4) raw QE files peptides 8742 proteins (including cell lines) no matched RNA-seq protein-level assembly MaxQuant Adromeda search Uniprot database VdB 95 samples (5 replicates) 95x15 raw Orbi. Velos files peptides 7526 proteins 86 matched RNA-seq protein & gene-level assembly IDPicker Myrimatch, MSGF+, Pepitome Refseq database
7 Q Exactive raw files Medical Oncology Data processing workflow Uniprot proteins MaxQuant IDPicker 3 Myrimatch, MSGF+ RefSeq version trypsin sequences MaxQuant text outputs Processed data, statistics & figures
8 Medical Oncology proteins FDR, 0.1% peptide FDR TCGA tissue proteins Amsterdam tissue proteins % OPL cell line proteins
9 Total spectral counts Medical Oncology
10 Unsupervised clustering of proteins with average count > 1.4 (4872) Medical Oncology
11 CCA O P VUmc L ONCO Proteomics Laboratory Medical Oncology Validation of CRC subtype markers using external TCGA CRC proteome data AMS-Connection TCGA Validation 1 Validation 2 is by profiling Match cohort CRC Rotterdam (planned) 11
12 Medical Oncology ccs1 vs ccs3, corrected p < 0.05 TCGA tissue 88 OPL tissue
13 Medical Oncology Examples CCS-subtype enriched markers CCS1-enriched CCS3-enriched Stroma protein part of OncoType Dx prognostic signature cells AMC TCGA cells AMC TCGA HPA: heterogeneous staining; Colon & CRC enriched Promising CRC subtype protein markers identified that link to prognostic groups 13
14 Medical Oncology Random Forrest algorithm 70-gene set training using Amsterdam tissues testing on TCGA prediction css1 css3 observed css css3 1 9
15 Medical Oncology Feature importance gene names
16 Medical Oncology Brute force, testing all 3-gene combinations Amsterdam training TCGA testing gene names 16
17 Medical Oncology Ongoing... Better normalization for cross dataset comparison Brute force approach with 4,5-gene combination, and sensitivity analysis Correlate mrna-protein in AMS and TCGA datasets Whole dataset Per CRC subtype Proteogenomics to search subtype-associated protein variants GO mining/ Network / pathway analysis to uncover CRC subtype associated biology and drug targets Selection of the most discriminatory subtype markers identified in both datasets for IHC follow-up
18 A bioinformatics pipeline for proteogenomics Wang X, et al. (2012) Protein identification using customized protein sequence databases derived from RNA-Seq data. J Proteome Re 11(2):
19 Q Exactive raw files Uniprot proteins new proteins MaxQuant Simple extention to the existing data processing workflow Proteogenomics MaxQuant text outputs Processed data, statistics & figures
20 ccle CCLE_hybrid_capture1650_hg19_NoCommonSNPs_NoNeutralVariants_CDS_ txt web ensembl Ensemble gtf Homo_sapiens.GRCh37.71.gtf web biomart + EntrezGene ID _biomart_export.txt ccle-1651.gtf web ensembl Homo_sapiens.GRCh37.71.dna.toplevel.fa.gz gffread -W ccle.fa Proteogenomics new proteins mutate six-frame translation
21 CCLE data: HT-29 EPHB4 mutation G A
22 Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal cancer RNA splicing accompanies tumor progression splicing translation premrna alternatively spliced mrna protein isoforms Tumor-specific protein isoforms could complement or outperform hemoglobin in CRC screening Design an approach to identify tumor specific protein variants Malgorzata A Komor et al. oral presentation at AACR 2016
23 Colorectal cancer (CRC) has a high cure rate when diagnosed early Population wide screening for colorectal cancer implemented in the Netherlands Fecal immunochemical test (FIT) Colonoscopy FIT+ FIT performance * Specificity: ~95% Sensitivity CRC : ~65% Sensitivity precursor lesions (advanced adenomas): ~27% Need for novel biomarkers * Van Veen W et al, Colorectal cancer screening: advice from the Health Council of the Netherlands, Ned.Tijdschr.Geneesk., 2009
24 Experimental design Down-modulation of splicing machinery to investigate differential splicing in a controlled setting RNA-seq Illumina HiSeq 2x125bp CRC cell lines SW480 Figure adapted from SF3B 1 proteomics LC-MS/MS QExactive PacBio Iso- Seq <10kb sisrsf1 siu2af1 sisf3b1 sint1 sint2 Splice site recognition: SRSF1, U2AF1 Proteogenomic pipeline
25 Quality and adapter trimming Reads mapping (STAR) Mapping QC (RseQC) Differential gene expression (DESeq2) Short RNAseq reads Sequenced full-length transcripts Reference annotation Differential splicing analysis (rmats) 3-frame translation Differential splice variants Potential protein variants Enriched protein database Identify MS/MS through database search Extract variant peptides Alternatively spliced proteins Mass spectra
26 Mass spectrometry revealed that some of the isoforms are translated into proteins Experiment sisf3b1 siu2af1 8 sisrsf1 7 Isoforms for which peptides where detected (PacBio) For a subset of proteins both isoform-specific and canonical peptides were detected Exon skipping: XXXXX ZZZZZ XXXXX------YYYY inclusion XXXXXYYYY exclusion XXXXXZZZZZ
27 Ongoing in proteogenomics The proteogenomic pipeline will be used for discovery and validation of CRC related splice variant proteins patient-derived 3D tissue cultures (organoids) human colorectal (tumor) tissues Validation of the diagnostic performance of these splice variant proteins in stool samples as a biomarker screening test
28 Discussions bioinformatics analysis regularly needs to access external resources and datasets raw data accessible pipeline reproducible cross dataset queries
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