1. DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health National Cancer Institute Frederick National Laboratory is a federally funded research and development center operated by SAIC-Frederick, Inc., for the National Cancer Institute. Hue Vuong Bioinformatics Analyst ABCC – Data Science and Information Technology Program March 8, 2016 Annotation, Visualization and Impact Analysis AVIA

2. Frederick National Laboratory for Cancer ResearchOutline Introduction to AVIA Input Types Impact Analysis Parameters Data Retrieval Visualization of Results and data Setup parameters for recurring submissions Protein/Gene Features annotation

3. Basic workflow for whole-exome and whole-genome sequencing projects. Stephan Pabinger et al. Brief Bioinform 2013;bib.bbs086 © The Author 2013. Published by Oxford University Press.

4. Frederick National Laboratory for Cancer Research Gene Regulation http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/Articleimage/201 5/MB/c5mb00310e/c5mb00310e-f1_hi-res.gif Methylation Promoters Structural Tf Binding Sites PTM Protein Binding Binding Sites Domains, etc Splicing miRNA

5. Overview of AVIA User submits request To AVIA website Request is logged in the AVIA website database (mySQL) User input data and parameters located on webserver User has mapped data WEBSERVER Annotation Email is sent to user to view reports on web. Reports can be downloaded Local Annot files Compute Servers

6. Frederick National Laboratory for Cancer Research AVIA – Annotation, Variation and Impact Analysis https://avia-abcc.ncifcrf.gov

7. Frederick National Laboratory for Cancer Research Intro to AVIA: Impact Analysis –Gene impact annotations from ANNOVAR framework http://www.openbioinformatics.org/annovar –Additional databases for annotating -Up to date annotations –Gene level annotations KEGG pathway maps with variant annotations Normal tissue expression Uniprot annotations –Structure –Variants –Domains –Visualizations and Prioritization

8. AVIA Formats – BED Header (optional) –Starts with ‘#’ Required Fields –Chromosome –Start position –Stop position –Reference Allele –Variant Allele #Chr StartStopRefAlleleVariant 20 14370 14370 G A 20 17330 17330 T A 20 1110696 1110696A G 20 1230237 1230237T A 20 1234567 1234570GTCT -

9. Frederick National Laboratory for Cancer Research AVIA Formats - VCF Header Arbitrary number of INFO defintion lines starting with ‘##’ Column definition line starts with ‘#’ Mandatory columns Chromosome (CHROM) Position of the Start of Variant (POS) Unique identifiers (ID) if none, then specify ‘.’ Reference Allele (REF) Comma separated list of alt alleles (ALT) Phred-scaled quality score (QUAL) Site filtering information (FILTER) User extensible annotation (INFO)

10. ##fileformat=VCFv4.0##fileformat=VCFv4.0 ##fileDate=20090805 ##source=myImputationProgramV3.1 ##reference=1000GenomesPilot-NCBI36 ##phasing=partial ##INFO= ##FILTER= ##FORMAT= #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT NA00001 NA00002 NA00003 20 14370 rs6054257 G A 29 PASS NS=3;DP=14;AF=0.5;DB;H2 GT:GQ:DP:HQ 0|0:48:1:51,51 1|0:48:8:51,51 1/1:43:5:.,. 20 17330. T A 3 q10 NS=3;DP=11;AF=0.017 GT:GQ:DP:HQ 0|0:49:3:58,50 0|1:3:5:65,3 0/0:41:3 20 1110696 rs6040355 A G,T 67 PASS NS=2;DP=10;AF=0.333,0.667;AA=T;DB GT:GQ:DP:HQ 1|2:21:6:23,27 2|1:2:0:18,2 2/2:35:4 20 1230237. T. 47 PASS NS=3;DP=13;AA=T GT:GQ:DP:HQ 0|0:54:7:56,60 0|0:48:4:51,51 0/0:61:2 20 1234567 microsat1 GTCT G,GTACT 50 PASS NS=3;DP=9;AA=G GT:GQ:DP 0/1:35:4 0/2:17:2 1/1:40:3

11. Frederick National Laboratory for Cancer Research AVIA Input Parameters: Data Format Input Formats BED-like VCF4 CLC Bio Ion Torrent Variant Caller HGVS* (Human Genome Variation Society) mRNA, cDNA, and Protein point Mutations ---------------------------------------------- Gene Symbols Input Format for Custom Database Queries Regions: Chr -> Start -> Stop -> FeatName -> Cutoff value (optional) Variants: Chr -> Start -> Stop -> Ref Allele -> Var Allele -> Annot * HGVS is still in beta testing

12. Frederick National Laboratory for Cancer Research Intro to AVIA: Analysis Options Genomic Workflows: Users can provide data for impact analysis Customized Feature Annotation & Visualization Basic Annotation (Gene only) Cascade annotations (filtering) miRNA SNP analysis

13. Frederick National Laboratory for Cancer Research Intro to AVIA: Analysis Options Protein Tools: Users can provide protein coordinates for impact analysis Annotate backwards from protein coordinates Visualize protein mutation on native protein structures e.g. BRAF:V600E

14. Frederick National Laboratory for Cancer Research Intro to AVIA: Analysis Options General Tools: Set up a configuration file Gene based converters File/Data converter tools Results Retrieval: Retrieve a result View sample results page

15. Frederick National Laboratory for Cancer Research Visualization using Circos* *Circos is currently unavailable

16. Frederick National Laboratory for Cancer Research Visualization* and Prioritization *Pathview is currently unavailable

17. Frederick National Laboratory for Cancer Research Links to other Visualizations CyPRUS GeneMania https://bioinfo-abcc.ncifcrf.gov/cyprus http://www.genemania.org/

18. DEMO

19. Frederick National Laboratory for Cancer Research Where to find more information Presentation Demo materials https://avia-abcc.ncifcrf.gov/apps/site/class Selected Annotation Review Comparison from Pabinger et al. https://avia- abcc.ncifcrf.gov/apps/site/viewPDF/?name=Pabinger_S7VariantAnnotation. pdf Database Information https://avia-abcc.ncifcrf.gov/apps/site/dbs Tutorial https://avia-abcc.ncifcrf.gov/apps/site/tutorials Contact or more information https://avia-abcc.ncifcrf.gov/apps/site/submit_a_question https://avia-abcc.ncifcrf.gov/apps/site/faq

20. Frederick National Laboratory for Cancer ResearchThanks ABCC Impact Analysis: Dr. Jack Collins Uma Mudunuri Dr. Brian Luke Dr. Sarangan Ravichandran Anney Che Please cite our paper: Vuong, H., et al. (2015) AVIA v2.0: annotation, visualization and impact analysis of genomic variants and genes, Bioinformatics (Oxford, England), 31, 2748-2750.