Ensembl Browser Workshop
- 1. Training materials Ensembl materials are protected by a CC BY license http://creativecommons.org/licenses/by/4.0/ If you wish to re-use these, please credit Ensembl for their creation If you use Ensembl for your work, please cite our papers http://www.ensembl.org/info/about/publications.html
- 2. Denise Carvalho-Silva European Molecular Biology Laboratory European Bioinformatics Institute Browsing Genes, Variation and Regulation data with Ensembl UCD - Dublin
- 3. Today 09:30-17:00 • Introduction to Ensembl • Browser walkthrough 10:45-11:00 coffee/tea • Browser exercises • BioMart (Talk + Exercises) 13:00-14:00 lunch break • Genetic variation (Talk + Exercises) 15:30-15:45 coffee/tea • Gene Regulation and/or Custom data (Talk + Exercises) • Wrap up, photo opportunity & feedback survey
- 5. Course Objectives What is Ensembl? What type of data can you get in Ensembl? How to navigate the Ensembl browser website? How to connect with Ensembl
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- 7. Introduction Why do we need/have genome browsers?
- 8. Genome sequencing 1977: 1st genome to be sequenced (5 kb) 2000: draft human sequence (3 gb) Large amounts of raw DNA sequence data
- 9. Raw DNA sequence data
- 11. Annotation of vertebrate genomeswww.ensembl.org pre.ensembl.org >80 genomes* D. melanogaster C. elegans S. cerevisae *Release 84 March 2016
- 12. 1 human genome à 3 assemblies www.ensembl.org grch37.ensembl.org e54.ensembl.org
- 13. EBI is an Outstation of the European Molecular Biology Laboratory. Comparative GenomicsGene models RegulationVariation Custom data displayProgrammatic access Toolkit Ensembl Features
- 14. EBI is an Outstation of the European Molecular Biology Laboratory. Comparative GenomicsGene models RegulationVariation Custom data displayProgrammatic access Toolkit Ensembl Features
- 16. Gene models in Ensembl Goal: Generate set of well-supported genes Automatic Manual
- 17. • many species • genome-wide at once • ~ 4 months • fewer species • gene by gene • many years Automatic and coding (20_) Manual and coding (00_) Automatic + Manual (“gold”) Manual and non-coding (00_) Automatic annotation* Manual annotation* * based on experimental, biological evidence (INSDC, UniProtKB…)
- 18. Ensembl genes & transcripts • merged annotation • higher confidence and quality • comprehensive: alternatively spliced transcripts UTR ExonIntron 5’ UTR3’ UTR Gold (identical annotation) = Automatic + Manual
- 19. Alternatively splicing rich and comprehensive annotation
- 20. Which transcript to use? http://www.ensembl.org/Help/Glossary?id=493 http://www.ensembl.org/Help/Glossary?id=492 APPRIS TSLs
- 21. CCDS project • annotate a consensus coding DNA sequence set • EBI, WTSI, UCSC and NCBI • Genome Res. 19:1316-23 (2009) http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi CCDS transcript
- 22. Disclaimer: which transcript to use No single method will tell us which transcript to use Decision on a case by case basis • All transcripts OR one/two well supported ones? List of transcripts: we offer choices based on • CCDS (Ensembl, HAVANA, NCBI, UCSC) • Golden transcripts (identical Ensembl and HAVANA) • Cross reference entries (e.g. UniProtKB, RefSeq) • APPRIS • TSLs
- 23. Annotation based on RNASeq http://www.ensembl.org/info/genome/genebuild/ rnaseq_annotation.html
- 25. Ensembl stable identifiers • ENSG########### Ensembl Gene ID • ENST########### Ensembl Transcript ID • ENSP########### Ensembl Peptide ID • ENSE########### Ensembl Exon ID • For non-human species a suffix is added: ENSMUSG MUS (Mus musculus) for mouse
- 27. Ensembl Browser Live demo: Walking through the website pages 11-31
- 28. The ESPN gene products are active in the inner ear, where it appears to play an essential role in normal hearing and balance. Let’s explore ESPN Before we start: background
- 29. A) What is the location and strand of the human ESPN gene? B) How can I view protein alignments and variants mapped to this location? C) Can I move data tracks up and down, share and delete tracks? Human ESPN: location
- 30. A) How can I find the genomic sequence of this gene? What is the ID of its first exon? B) Can I display the genomic coordinates and variants on this sequence? C) Can I find information on the expression of this gene in different tissues? Human ESPN: gene
- 31. A) How many exons does the longest ESPN transcript have? Are there any completely untranslated exons? B) Can I find its cDNA sequence? C) What are the UniProt and RefSeq entries cross referenced to this transcript? Human ESPN: transcript
- 32. Ensembl Browser Exercises pages 32-35 Answers www.ebi.ac.uk/~denise/workshops/2016/ dublin/answers Feel free to explore your favourite gene/region too!
- 33. EBI is an Outsta,on of the European Molecular Biology Laboratory. BioMart
- 34. Outline • Definitions • The principle: 4 steps • Tutorial: simple query in human • Find Ensembl BioMart and BioMart elsewhere • Sophisticated platforms: mart services, APIs, etc… • Exercises
- 35. Would you like to… • … convert protein IDs into gene IDs or names? • … get a list of all genes mapped to a region deleted in a patients’ cohort? • … export sequences for a bunch of genes or variants? If you answered yes, keep listening!
- 36. What is BioMart? • Free service for easy retrieval of Ensembl data • Data export tool with little/no programming required • Complex queries with a few mouse clicks • Output formats (.xls, .csv, fasta, tsv, html)
- 37. The four-step principle DATA FILTERS ATTRIBUTES RESULTS IDs Regions Domains Expression Tables Fasta Dataset Database Homologs Sequences Features Structures
- 38. Choosing the data Database and dataset
- 39. Limit your data set (information that you know) Selecting the filters Click “Count” to see if BioMart is reading the input data
- 40. Picking the attributes Determine output columns (information you want to know)
- 42. Getting the results Tables/sequences click “Unique results only” For the full table: click View “ALL” rows or “Go”
- 43. Selected IBD genes IL23R, PTPN22, CUL2, C1orf106, IL18RAP
- 44. For the IL23R, PTPN22, CUL2, C1orf106 and IL18RAP genes, use BioMart to retrieve a table (.xls) containing: • Associated gene name, ENSG and ENST IDs • Chromosome name, gene start and end • GO term name and Interpro description Tutorial: BioMart
- 45. The four-step principle DATA FILTERS ATTRIBUTES RESULTS Gene Gene name, ENSG/ENST ID, Chr start end, GO term name, Interpro description .xls table Human IL23R, CUL2, PTPN22, C1orf106 IL18RAP
- 48. Ensembl BioMarts
- 50. More sophisticated platforms • BioMart queries: MartService www.biomart.org/martservice.html • APIs: PERL, Java, Web Services • Third party softwares galaxyproject.orgbioconductor.orgtaverna.org.uk
- 53. BioMart Step-by-step example pages 36-40 Exercises pages 41-43 Answers www.ebi.ac.uk/~denise/workshops/2016/ dublin/answers Feel free to explore BioMart in other contexts too!
- 54. EBI is an Outsta,on of the European Molecular Biology Laboratory. Genetic Variation
- 55. Outline • Classes of variation, species and sources • Browsing variation data: some entry points Location tab Gene tab Variation tab • Phenotypic data and population genetics • How to annotate your own variants • Exercises
- 56. 1) Large scale: structural (> 50 base pairs) Genetic variation duplication deletion inversion translocation loss 2) Short scale: SNPs (or SNVs), indels G A C TG A C TA T C G GG GTT TCC CA A A G A A TG A C TT T C G G- G-T TCC -A A A
- 57. Species with variation data Understand the types of genetic variation data and how to view them in the context of our genomes
- 58. Sources of variation data • Import alleles and frequencies • Annotate variants http://www.ensembl.org/info/docs/variation/sources_documentation.html
- 59. Location tab: across a region SVsSNPs Ensembl genes
- 61. Variation tab: variant centric summary data SNP or SV
- 62. Variants on the karyotype
- 63. Phenotype data in Ensembl species and sources
- 64. Population data for variants http://hapmap.ncbi.nlm.nih.gov/ http://www.1000genomes.org
- 65. pie charts: 1KG super populations Human Population Genetics
- 66. Coffee intake is a worldwide phenomenon with Finland at the top, and UK in the 44th place. Is caffeine consumption in our genes? A) What are the chromosome locations of variants associated with this phenotype? B) Which variant has got the most significant association? C) What is the ancestral allele of this variant? Is it conserved in eutherian mammals? D) What is the most frequent allele in GBR? E) Can you download this variant and 200 nt upstream and downstream flanking sequence in RTF (Rich Text Format)? Live demo
- 67. before we find out • What is it? • What does it do? • Where can I find it?
- 68. I’ve got a list of genetic variants from my resequencing project of a cohort study of breast cancer in London. The positions are all on chromosome 9, GRCh37 assembly: 131090740 A/- (positive strand) 131084628 C/A (positive strand) 131085358 C/G (positive strand) 131085196 G/A (positive strand) 1) Do any of these cause a change at the amino acid level? 2) Are these predicted to be deleterious according to PolyPhen? 3) Can I get the flanking sequence (200 nt both up and downstream) for the known variants in this set? A use case: cancer patients
- 69. My resequencing experiments cancer patients X healthy controls 9 131090740 131090740 A/- 9 131084628 131084628 C/A 9 131085358 131085358 C/G 9 131085196 131085196 G/A Chromosome Alleles End Start Positions in the genome vary between the two groups
- 70. Can I annotate these variants? • Variant Effect Predictor • Annotate variants (SNPs, CNVs, indels) • Available for GRCh38 and GRCh37 (hg=19) Yes, you can! PMID: 20562413 Perl script Web interface REST API XML
- 71. CODING Synonymous INTRONIC5’ UTR ATG AAAAAAA Regulatory Splice sites CODING Missense 3’ UTR5’ Upstream 3’ downstream Mapping variants on transcripts Identify transcripts that overlap variants and predict the consequence of these on Ensembl (or RefSeq) transcripts using
- 72. Consequence terms for variants http://www.ensembl.org/info/genome/variation/predicted_data.html#consequence_type_table * defined by the Sequence Ontology (SO) project (http://www.sequenceontology.org/)
- 73. SIFT sift.jcvi.org/ Consequence: missense GAG >GGG Glu > Gly PolyPhen-2 genetics.bwh.harvard.edu/pph2/ Condel dbNSFP
- 76. Output options in GAG > GGG Glu > Gly GAG > GAA Glu > Glu
- 77. Queued Running Done Failed Save to your account (log in) Edit and resubmit your job Delete job Ticket system in Ticket identifier Job name
- 78. Viewing results SO consequence terms* *http://www.sequenceontology.org/index.html
- 79. ensembl.org/info/docs/tools/vep/online/results.html#summary Table • Before / after filtering • novel / existing variants Pie charts (consequence terms) • total observed (more than one per variant) • Separate chart: coding consequences Viewing results
- 80. Navigate results (one row per variant/ transcript overlap) Show/hide columns in results table more columns: scroll right • Download results • Send results to BioMart Create and edit filters ensembl.org/info/docs/tools/vep/online/results.html#table results table
- 81. Filters consist of three components Field • e.g. Consequence, biotype Operator • e.g. is, matches (partial string matches) Value • the value to compare against • some fields have autocomplete values Multiple filters allowed with logical relationship (AND, OR) Active filters can be edited too! ensembl.org/info/docs/tools/vep/online/results.html#filter Filtering results
- 82. My resequencing experiments cancer patients X healthy controls 9 131090740 131090740 A/- 9 131084628 131084628 C/A 9 131085358 131085358 C/G 9 131085196 131085196 G/A Chromosome Alleles End Start Positions in the genome vary between the two groups
- 85. Things to bear in mind 1) No distinction between polymorphisms and mutations. Exception HGMD and COSMIC: all mutations; 2) C/T à first allele is the one in the reference genome, not necessarily the major or the ancestral; 3) Ensembl reports all alleles on the forward strand (different from dbSNP).
- 86. Ensembl Variation API Variation Schema Description http://useast.ensembl.org/info/docs/api/variation/index.html
- 89. Ensembl Genetic Variation Exercises pages 46-50 Answers www.ebi.ac.uk/~denise/workshops/2016/ dublin/answers Feel free to explore your favourite variant/phenotype too!
- 90. EBI is an Outsta,on of the European Molecular Biology Laboratory. Gene Regulation
- 91. Outline • Definition and models • Epigenetics and Epigenomics • Ensembl Regulation: goal, data sources, species • Viewing / accessing regulation data in Ensembl • Track hubs: ENCODE, Blueprint • Exercises
- 92. Regulation of gene expression • Change in the production of mRNA/proteins ( or ) • From the transcription to post-translational levels • Models of regulation of gene transcription • Basic • Expanded • Complete ??
- 93. Transcription regulation Transcription Factor Binding Sites Promoter Gene mRNA Transcription Factors Activation Repression RNA polymerase complex 2 nm basic model • TF binding (promoters, enhancers) à transcription
- 94. Nucleosomes Histones Histone marks CpG methylation 11 nm Transcription regulation expanded model • Epigenetic marks may affect the binding of TFs
- 95. Histone modifications dynamically regulating genes JillS.Butler,andSharonY.R.Dent Blood2013;121:3076-3084
- 96. Packed Chromatin 30 nm Open Chromatin Distal enhancer Complete (???) model Transcription regulation
- 97. Epigenetics/Epigenomics Epigenetics* The study of inherited changes in phenotype without changes in genotype Epigenomics Epigenetics on a genome-wide scale http://integratedhealthcare.eu/ *One of the routes to regulate gene transcription
- 98. Measuring gene expression Northern/Western blot Microarrays SAGE AdpatedfromDarrylLeja,IanDunham NGS techniques DNase-seq ChIP-seq RNASeq RT-qPCR
- 99. ChIP-sequencing crosslink and shear TF1 TF2 TF3 TF1 TF3TF2 Antibodies and IP unlink, purify and DNA sequencing Y YY TF1 TF3 TF2 ACGTC CGCTT GAACA map back to the genome DNA and proteins
- 100. Ensembl Regulation Goal: Annotate the genome with features that may play a role in the transcriptional regulation of genes Multiple data sources: collection and summary http://www.ensembl.org/info/docs/funcgen/regulation_sources.html http://www.ensembl.org/Homo_sapiens/Experiment/
- 101. Data source: ENCODE “Encyclopedia of DNA Elements” Trying to assign function to many regions as possible Transcription and regulatory information 4,626 datasets, 2,498 cell types à functional elements PMID: 22955616, PMID: 17571346 http://www.nature.com/encode/#/threads
- 102. Data source: Roadmap NIH consortium: public resource of normal epigenomes DNA methylation, histone marks, open chromatin, small RNA http://www.roadmapepigenomics.org/data http://www.roadmapepigenomics.org/publications
- 103. • EU consortium: generate 100 reference epigenomes • Blood cells: healthy individuals and malignant leukaemic counterparts • 1046 experiments {ChIP, RNA, Bisulfite, DNase}-Seq • 425 cell types and seven cell lines • http://www.blueprint-epigenome.eu/ Data source: Blueprint
- 104. Dataset for the Ensembl build raw data à Ensembl Regulation pipeline à Ensembl annotation
- 105. Regulation data: view MultiCell: all cell lines combined Displayed by default
- 106. Regulatory features: view Configure this page à Regulation à Regulatory features For single and individual cell lines, e.g. GM12878, HUVEC
- 107. ChiP-Seq signal for TF signal Regulatory Features: motifs Ensembl regulatory feature Position Weight Matrix for TF (JASPAR database)
- 108. Viewing the raw NGS data DNaseI and TFBS Histone marks and polymerases Configure this page à Regulation à Open chromatin &… Configure this page à Regulation à Histones &…
- 109. How to choose raw data: matrix Supporting evidence: 1) Open chromatin & TFBS 2) Histones & polymerases http://tinyurl.com/matrix-ensembl
- 110. CTCF enriched Predicted Weak Enhancer/Cis-‐reg element Predicted Transcribed Region Predicted Enhancer Predicted Promoter Flank Predicted Repressed/Low AcAvity Predicted Promoter with TSS Segmentation data in Ensemblcategoriesof combinedsegments Configure this page à Regulation à Regulatory features
- 111. Experimental confirmation • CTCF: good recall, reproducible across multiple cell lines, tight boundaries. • TSS: • 88.9% of FANTOM 5 strict TSSs were covered. • Enhancers: • 92.4% of 882 VISTA enhancers were detected. • 80.3% of 40279 robust FANTOM 5 enhancers were found.
- 112. Methylation data in Ensembl CpG DNA methylation (RRBS, WGBS, MeDIP) ENCODE and PMID: 18577705 Configure this page à Regulation à DNA Methylation
- 113. The STRADA controls tumor suppressor activities of LKB1 (https://www.wikigenes.org/) A. What are the Ensembl regulatory features annotated in this gene? B. Are there any features in the 5’ region of STRADA? C. Do the regulatory features for K562, CD8+ cells (ENCODE) and erythroblast (Blueprint) differ at this region? D. What is the stable IDs of the most 5’ regulatory feature? Tutorial
- 115. Things to bear in mind 1) The annotation of regulatory elements in Ensembl highlight where the biochemical data (ChIP-seq, etc) maps to on the human (mouse) genomes; 2) Features can be nearby genes but might not affect their transcription/expression; 3) Disclaimer: Ensembl can not tell you how your favourite gene is regulated.
- 116. In addition to the big names CpG islands, TSS, miRNA target predictions (TarBase) Configure this page à Regulation à Other regulatory regions Configure this page à Sequence and assembly à Simple features
- 117. Track hubs in Ensembl
- 118. ENCODE data hub in Ensembl www.ensembl.org/info/encode.html >2,800 data tracks
- 119. Ensembl Regulation in BioMart Human, mouse and fruit fly FILTERS ATTRIBUTES
- 120. Ensembl Regulation API http://useast.ensembl.org/info/docs/api/funcgen/index.html Funcgen Schema Description
- 123. Ensembl Regulation Exercises Pages 52-54 Answers www.ebi.ac.uk/~denise/workshops/2016/ dublin/answers Feel free to explore your favourite gene/genomic region!
- 124. EBI is an Outsta,on of the European Molecular Biology Laboratory. Custom data display
- 125. Outline • Overview • Supported file formats • Add your own data • Where to view your own data • Tutorials and exercises
- 126. Overview • Genome browsers have pre-defined sets of data • Need to display personal data • Compare one’s own data to publicly available one • Requisite: own data organised to specific rules http://www.ensembl.org/info/website/upload/index.html
- 127. Supported files in Ensembl Sequence alignments http://www.ensembl.org/info/website/upload/index.html#formats • BAM (compact representation) CRAM (compressed version) Flexible definition of data lines Variation data Feature information Continuous-valued data (probability scores) • VCF: Variant Call Format • BED (Browser Extensible Data) e.g. chr, start, end • Gene Finding Format (GFF) General Transfer Format (GTF) • Wig, BigWig
- 128. Add custom data • Data upload: small files (< 5MB; file name or URL) • Attach your data: larger files (> 5MB; URL) Things to bear in mind Saved in a temp location (file system) Saved in a db if logged in Standard security http, https or ftp
- 129. How can I add my data
- 130. Where to view my data
- 131. Structural variants in the 350-50 kb region upstream of the SOX9 cause severe dysplasia and other phenotypes. Many enhancers (e.g. E250, located at -250 kb) activate the SOX9 promoter, whereas E70 seems to be active in somatic tissues. CGH/other experiments have revealed the following deletions : 17 69872078 69886644 patient1 17 70040357 70049956 patient2 17 70111957 70116270 patient3 A) Is any of these deletions known to be polymorphic in 1KG? B) Would these deletions affect E250 and E70? C) Do they map to regions of promoter activity or CpG islands? Tutorial
- 132. Custom data display video http://tinyurl.com/ensembl-upload
- 134. Custom data display Exercises Pages 55-57 Answers www.ebi.ac.uk/~denise/workshops/2016/ dublin/answers Feel free to attach your own data
- 135. Wrap up Ensembl is the place! Genes, genomes, variants, regulatory features, tools and more
- 136. Ensembl Retreat June 2015
- 137. Latest publication
- 138. Acknowledgements The Entire Ensembl Team Funding Co-funded by the European Union
- 139. Your take home message
- 142. Training materials Ensembl materials are protected by a CC BY license http://creativecommons.org/licenses/by/4.0/ If you wish to re-use these, please credit Ensembl for their creation If you use Ensembl for your work, please cite our papers http://www.ensembl.org/info/about/publications.html