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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
Denise Carvalho-Silva European Molecular Biology Laboratory
European Bioinformatics Institute
Browsing Genes, Variation and Regulation data with Ensembl
UCD - Dublin
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
http://www.ebi.ac.uk/ ~denise/workshops/
2016/dublin
Materials
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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Introduction Why do we need/have genome browsers?
Genome sequencing
1977: 1st genome to be sequenced (5 kb) 2000: draft human sequence (3 gb)
Large amounts of raw DNA sequence data
Raw DNA sequence data
Annotation: making sense
Annotation of vertebrate genomes
w
ww
.ens
embl
.org
pre.
ense
mbl
.org
>80 genomes* D. melanogaster
C. elegans S. cerevisae
*Release 84 March 2016
1 human genome à 3 assemblies
www.ensembl.org grch37.ensembl.org
e54.ensembl.org
EBI is an Outstation of the European Molecular Biology Laboratory.
Comparative Genomics Gene models
Regulation Variation
Custom data display Programmatic access
Toolkit
Ensembl Features
EBI is an Outstation of the European Molecular Biology Laboratory.
Comparative Genomics Gene models
Regulation Variation
Custom data display Programmatic access
Toolkit
Ensembl Features
Ense
mb
l au
tom
atic
an
not
atio
n
Gene models in Ensembl
Goal: Generate set of well-supported genes
Automatic Manual
• 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…)
Ensembl genes & transcripts
• merged annotation
• higher confidence and quality
• comprehensive: alternatively spliced transcripts
UTR Exon Intron
5’ UTR 3’ UTR
Gold (identical annotation) = Automatic + Manual
Alternatively splicing
rich and comprehensive annotation
Which transcript to use?
http://www.ensembl.org/Help/Glossary?id=493 http://www.ensembl.org/Help/Glossary?id=492
APPRIS
TSLs
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
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
Annotation based on RNASeq
http://www.ensembl.org/info/genome/genebuild/rnaseq_annotation.html
ncRNA gene annotation
http://www.ensembl.org/info/genome/genebuild/ncrna.html
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
Ensembl Browser
Live demo: Walking through the website
pages 11-31
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
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
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
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
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!
EBI is an Outsta,on of the European Molecular Biology Laboratory.
BioMart
Outline
• Definitions
• The principle: 4 steps • Tutorial: simple query in human
• Find Ensembl BioMart and BioMart elsewhere
• Sophisticated platforms: mart services, APIs, etc… • Exercises
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!
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)
The four-step principle
DATA FILTERS ATTRIBUTES
RESULTS
IDs Regions Domains
Expression
Tables Fasta
Dataset
Database Homologs Sequences Features
Structures
Choosing the data
Database and dataset
Limit your data set (information that you know)
Selecting the filters
Click “Count” to see if BioMart is reading
the input data
Picking the attributes
Determine output columns (information you want to know)
The different attributes
Getting the results
Tables/sequences
click “Unique results only”
For the full table: click View “ALL”
rows or “Go”
Selected IBD genes
IL23R, PTPN22, CUL2, C1orf106, IL18RAP
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
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
Ensembl BioMart
Live demo
Find BioMart
www.ensembl.org/biomart
Ensembl BioMarts
http://tinyurl.com/biomart-video
BioMart video
More sophisticated platforms
• BioMart queries: MartService www.biomart.org/martservice.html
• APIs: PERL, Java, Web Services
• Third party softwares
galaxyproject.org bioconductor.org taverna.org.uk
Ensembl BioMart
Thomas Maurel
Amonida Zadissa
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!
EBI is an Outsta,on of the European Molecular Biology Laboratory.
Genetic Variation
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
1) Large scale: structural (> 50 base pairs)
Genetic variation
duplication deletion inversion translocation loss
2) Short scale: SNPs (or SNVs), indels
G A C T G A C T A T C G G G G T T T C C C A A A
G A A T G A C T T T C G G - G - T T C C - A A A
Species with variation data
Understand the types of genetic variation data and how to view them in the context of our genomes
Sources of variation data
• Import alleles and frequencies
• Annotate variants
http://www.ensembl.org/info/docs/variation/sources_documentation.html
Location tab: across a region
SVs SNPs
Ensembl genes
Gene tab: gene-centric SNPs
SVs
Variation tab: variant centric
summary data
SNP or SV
Variants on the karyotype
Phenotype data in Ensembl species and sources
Population data for variants
http://hapmap.ncbi.nlm.nih.gov/
http://www.1000genomes.org
pie charts: 1KG super populations
Human Population Genetics
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
before we find out
• What is it?
• What does it do?
• Where can I find it?
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
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
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
CODING Synonymous
INTRONIC 5’ UTR
ATG AAAAAAA Regulatory
Splice sites
CODING Missense
3’ UTR 5’ Upstream 3’ downstream
Mapping variants on transcripts
Identify transcripts that overlap variants and predict the consequence of these on Ensembl (or RefSeq) transcripts using
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/)
SIFT sift.jcvi.org/
Consequence: missense GAG >GGG Glu > Gly
PolyPhen-2 genetics.bwh.harvard.edu/pph2/ Condel
dbNSFP
Ensembl tools http://www.ensembl.org/tools.html
http://www.ensembl.org/vep
Inputting data into
Chromosome Start End Alleles Strand
Output options in
GAG > GGG Glu > Gly
GAG > GAA Glu > Glu
Queued Running Done Failed
Save to your account (log in) Edit and resubmit your job Delete job
Ticket system in
Ticket identifier Job name
Viewing results
SO consequence terms*
*http://www.sequenceontology.org/index.html
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
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
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
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
Ensembl VEP
Live demo
VEP video
http://tinyurl.com/vep-video
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).
Ensembl Variation API
Variation Schema Description
http://useast.ensembl.org/info/docs/api/variation/index.html
Variation Team
Fiona Cunningham
Will McLaren
Laurent Gil
Sarah Hunt
Anja Thormann
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!
EBI is an Outsta,on of the European Molecular Biology Laboratory.
Gene Regulation
Outline • Definition and models
• Epigenetics and Epigenomics
• Ensembl Regulation: goal, data sources, species • Viewing / accessing regulation data in Ensembl
• Track hubs: ENCODE, Blueprint • Exercises
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 ??
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
Nucleosomes
Histones
Histone marks
CpG methylation
11 nm
Transcription regulation expanded model
• Epigenetic marks may affect the binding of TFs
Histone modifications dynamically regulating genes
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Packed Chromatin
30 nm
Open Chromatin
Distal enhancer
Complete (???) model
Transcription regulation
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
Measuring gene expression
Northern/Western blot Microarrays
SAGE
Adp
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NGS techniques
DNase-seq ChIP-seq RNASeq
RT-qPCR
ChIP-sequencing
crosslink and shear
TF1 TF2 TF3
TF1 TF3 TF2
Antibodies and IP
unlink, purify and DNA sequencing
Y Y Y
TF1 TF3 TF2
ACGTC CGCTT GAACA
map back to the genome
DNA and proteins
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/
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
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
• 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
Dataset for the Ensembl build
raw data à Ensembl Regulation pipeline à Ensembl annotation
Regulation data: view
MultiCell: all cell lines combined Displayed by default
Regulatory features: view
Configure this page à Regulation à Regulatory features
For single and individual cell lines, e.g. GM12878, HUVEC
ChiP-Seq signal for TF
signal
Regulatory Features: motifs
Ensembl regulatory feature
Position Weight Matrix for TF (JASPAR database)
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 &…
How to choose raw data: matrix
Supporting evidence: 1) Open chromatin & TFBS 2) Histones & polymerases
http://tinyurl.com/matrix-ensembl
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 Ensembl ca
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Configure this page à Regulation à Regulatory features
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.
Methylation data in Ensembl CpG DNA methylation (RRBS, WGBS, MeDIP)
ENCODE and PMID: 18577705
Configure this page à Regulation à DNA Methylation
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
Browsing Regulation data
tinyurl.com/ensembl-regulation
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.
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
Track hubs in Ensembl
ENCODE data hub in Ensembl www.ensembl.org/info/encode.html
>2,800 data tracks
Ensembl Regulation in BioMart
Human, mouse and fruit fly
FILTERS
ATTRIBUTES
Ensembl Regulation API
http://useast.ensembl.org/info/docs/api/funcgen/index.html
Funcgen Schema Description
Regulation Team
Thomas Juettemann
Myrto Kostadima
Ilias Lavidas
Michael Nuhn
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!
EBI is an Outsta,on of the European Molecular Biology Laboratory.
Custom data display
Outline • Overview
• Supported file formats
• Add your own data
• Where to view your own data
• Tutorials and exercises
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
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
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
How can I add my data
Where to view my data
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
Custom data display video
http://tinyurl.com/ensembl-upload
Custom data display
Exercises Pages 55-57
Answers
www.ebi.ac.uk/~denise/workshops/2016/ dublin/answers
Feel free to attach your own data
Wrap up Ensembl is the place!
Genes, genomes, variants, regulatory features, tools and more
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