EBI is an Outstation of the European Molecular Biology Laboratory.

Ensembl Tools

Questions?

• We’ve muted all the mics• Ask questions in the Chat box in

the webinar interface• I will check the Chat box

periodically for questions• There’s no threading so please

respond with @name

Objectives

• What is Ensembl?

• What tools are available in Ensembl?

• How to use the online tools in Ensembl.

• Where to go for help and documentation.

Overview

• Introduction to Ensembl

• BLAST/BLAT

• Sequence searching

• Assembly Converter

• Convert files between genome assemblies

• Data Slicer

• Pull out sections of VCF and BAM files

• File Chameleon

• Custom download of reference files for NGS analysis

• Variant Effect Predictor (VEP)

• Analyse your own variants

Introduction

Why do we need genome browsers?

1977: 1st genome to be sequenced (5 kb)

2004: finished human sequence (3 Gb)

CGGCCTTTGGGCTCCGCCTTCAGCTCAAGACTTAACTTCCCTCCCAGCTGTCCCAGATGACGCCATCTGAAATTTCTTGGAAACACGATCACTTTAACGGAATATTGCTGTTTTGGGGAAGTGTTTTACAGCTGCTGGGCACGCTGTATTTGCCTTACTTAAGCCCCTGGTAATTGCTGTATTCCGAAGACATGCTGATGGGAATTACCAGGCGGCGTTGGTCTCTAACTGGAGCCCTCTGTCCCCACTAGCCACGCGTCACTGGTTAGCGTGATTGAAACTAAATCGTATGAAAATCCTCTTCTCTAGTCGCACTAGCCACGTTTCGAGTGCTTAATGTGGCTAGTGGCACCGGTTTGGACAGCACAGCTGTAAAATGTTCCCATCCTCACAGTAAGCTGTTACCGTTCCAGGAGATGGGACTGAATTAGAATTCAAACAAATTTTCCAGCGCTTCTGAGTTTTACCTCAGTCACATAATAAGGAATGCATCCCTGTGTAAGTGCATTTTGGTCTTCTGTTTTGCAGACTTATTTACCAAGCATTGGAGGAATATCGTAGGTAAAAATGCCTATTGGATCCAAAGAGAGGCCAACATTTTTTGAAATTTTTAAGACACGCTGCAACAAAGCAGGTATTGACAAATTTTATATAACTTTATAAATTACACCGAGAAAGTGTTTTCTAAAAAATGCTTGCTAAAAACCCAGTACGTCACAGTGTTGCTTAGAACCATAAACTGTTCCTTATGTGTGTATAAATCCAGTTAACAACATAATCATCGTTTGCAGGTTAACCACATGATAAATATAGAACGTCTAGTGGATAAAGAGGAAACTGGCCCCTTGACTAGCAGTAGGAACAATTACTAACAAATCAGAAGCATTAATGTTACTTTATGGCAGAAGTTGTCCAACTTTTTGGTTTCAGTACTCCTTATACTCTTAAAAATGATCTAGGACCCCCGGAGTGCTTTTGTTTATGTAGCTTACCATATTAGAAATTTAAAACTAAGAATTTAAGGCTGGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCGGATCACTTGAGGCCAGAAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCCTATCTCTACTAAAAATACAAAAAATGTGCTGCGTGTGGTGGTGCGTGCCTGTAATCCCAGCTACACGGGAGGTGGAGGCAGGAGAATCGCTTGAACCCTGGAGGCAGAGGTTGCAGTGAGCCAAGATCATGCCACTGCACTCTAGCCTGGGCCACATAGCATGACTCTGTCTCAAAACAAACAAACAAACAAAAAACTAAGAATTTAAAGTTAATTTACTTAAAAATAATGAAAGCTAACCCATTGCATATTATCACAACATTCTTAGGAAAAATAACTTTTTGAAAACAAGTGAGTGGAATAGTTTTTACATTTTTGCAGTTCTCTTTAATGTCTGGCTAAATAGAGATAGCTGGATTCACTTATCTGTGTCTAATCTGTTATTTTGGTAGAAGTATGTGAAAAAAAATTAACCTCACGTTGAAAAAAGGAATATTTTAATAGTTTTCAGTTACTTTTTGGTATTTTTCCTTGTACTTTGCATAGATTTTTCAAAGATCTAATAGATATACCATAGGTCTTTCCCATGTCGCAACATCATGCAGTGATTATTTGGAAGATAGTGGTGTTCTGAATTATACAAAGTTTCCAAATATTGATAAATTGCATTAAACTATTTTAAAAATCTCATTCATTAATACCACCATGGATGTCAGAAAAGTCTTTTAAGATTGGGTAGAAATGAGCCACTGGAAATTCTAATTTTCATTTGAAAGTTCACATTTTGTCATTGACAACAAACTGTTTTCCTTGCAGCAACAAGATCACTTCATTGATTTGTGAGAAAATGTCTACCAAATTATTTAAGTTGAAATAACTTTGTCAGCTGTTCTTTCAAGTAAAAATGACTTTTCATTGAAAAAATTGCTTGTTCAGATCACAGCTCAACATGAGTGCTTTTCTAGGCAGTATTGTACTTCAGTATGCAGAAGTGCTTTATGTATGCTTCCTATTTTGTCAGAGATTATTAAAAGAAGTGCTAAAGCATTGAGCTTCGAAATTAATTTTTACTGCTTCATTAGGACATTCTTACATTAAACTGGCATTATTATTACTATTATTTTTAACAAGGACACTCAGTGGTAAGGAATATAATGGCTACTAGTATTAGTTTGGTGCCACTGCCATAACTCATGCAAATGTGCCAGCAGTTTTACCCAGCATCATCTTTGCACTGTTGATACAAATGTCAACATCATGAAAAAGGGTTGAAAAAAGGAATATTTTAATAGTTTTCAGTTACTTT

We need to make the data mean something…

http://www. ensembl.org

http://www.ncbi.nlm.nih.gov/mapview

http://genome.ucsc.edu

Ensembl Features

• Gene builds for ~70 species

• Gene trees

• Regulatory build (ENCODE)

• Variation display and VEP

• Display of user data

• BioMart (data export)

• Programmatic access via the APIs

• Completely Open Source

Access scales

Whole genome

Groups

One by oneMain browserMobile site

BioMartREST APIVEP

Perl APIMySQL

FTP

Vertebrate species on Ensembl

Image obtained using Dendroscope:

Dendroscope 3: An interactive tool for rooted phylogenetic trees and networks

D.H. Huson and C ScornavaccaSystematic Biology, 2012

Non-vertebrates on Ensembl genomes

FungiBacteria

Plants

Protists

Metazoa

www.ensemblgenomes.org

Ensembl and Ensembl GenomesEnsembl EnsemblGenomes

Released 2000 2009

Species Vertebrates (fly, worm and yeast as outgroups)

Non-vertebrates (protists, plants, fungi, metazoa, bacteria)

Annotation by Ensembl in collaboration with the scientific communities

URL www.ensembl.org www.ensemblgenomes.org

Release cycle

89May 2017

2-3 months

New genome assemblies

Updated variation

data

Updated regulation

data

New/updated interfaces

Updated gene sets

Compara on new genes and genomes

Underlying software updates

90July 2017

Ensembl Tools

Tools allow:

• Interpretation and processing of your own data• Custom download of Ensembl data for further

analysis

BLAST/BLAT for sequence searching

• Find Ensembl sequences that match your sequence using BLAST/BLAT

• Search:• Nucleotide sequences• Protein sequences• Short sequences (eg primers, morpholinos, siRNAs)

• Search against• Genomic sequences• cDNA sequences• Protein sequences

Hands on – BLAST/BLAT

• I’ve designed a pair of primers for RT-PCR against human BRCA2

• I want to make sure they don’t have any non-specific hits that will mess up my RT-PCR results

• The sequences are:

>fwdGAGGACTCCTTATGTCCAAATTT

>revGAGAATCAGCTTCTGGGGTAATAA

Assembly converter

• You have data mapped to an old genome assembly• You want to update your data to map it to a new one

What is a genome assembly?

CGGCCTTTGGGCTCCGCCTTCAGCTCAAGATCCGCCTTCAGCTCAAGACTTAACTTC

GGGCTCCGCCTTCAGCTC ACTTAACTTCCCTCCCAGCTGTCC

AACTTCCCTCCCAGCTTCCCAGCTGTCCCAGATGACGCCATC

CAGATGACGCC

CAGCTGTCCCAGATGACCGGCCTTTGGGCTCC

CGGCCTTTGGGCTCCGCCTTCAGCTCAAGACTTAACTTCCCTCCCAGCTGTCCCAGATGACGCCATC

Sequence reads

Match up overlaps

Genome assembly

CGGCCTTTGGGCTCCGCCTTCAGCTCAAGA

TCCGCCTTCAGCTCAAGACTTAACTTC

GGGCTCCGCCTTCAGCTC

ACTTAACTTCCCTCCCAGCTGTCC

AACTTCCCTCCCAGCTTCCCAGCTGTCCCAGATGACGCCATC

CAGATGACGCC

CAGCTGTCCCAGATGAC

CGGCCTTTGGGCTCC

Genome contigs

CM

IM

AL

BL

BL102

AL476

CM

553IM

768

Reference alleles

IM

CM

AL

BL

BL102

AL476

CM

553IM

768

BL102

AGTCGTAGCTAGCTAGGCCATAGGCGA

Frequency T = 0.05, frequency G = 0.95G is the allele in all primatesT causes disease susceptibility

Perhaps G should be the reference allele?We can replace the region with a new contig

Genome Gaps

IM

CM

AL

BL

BL102

AL476

CM

553IM

768

BL102

AL476

Gap in the genome caused by:● Poor sequencing at this

region● No contig was ever

cloned

We can fill in the gap with a new contig

Incorrectly assembled contigs

IM

CM

AL

BL

BL102

AL476

CM

553IM

768

CM

AL

BL

BL102

AL476

CM

553IM

768

IM

New genome assemblies

• Fixing errors in the genome produces a new genome assembly

• New genome assemblies mean re-mapping of all genome features

• Ensembl will stop updating the old assembly when a new one is brought in

• You’ve got data mapped to the old assembly and you want to compare to the up-to-date Ensembl annotation

Assembly converter

• Converts genome coordinates to a different genome assembly.

• Works with:• BED (simple coordinates)• GFF (gene, transcript and exon coordinates)• GTF (gene, transcript and exon coordinates)• WIG (values plotted against the genome)• VCF (variants)

Hands-on – Assembly converter

• We’re going to convert a small BED file from the human genome assembly GRCh37 to the more recent GRCh38

• BED is a simple features format which lists the start and end coordinate of the feature.

5 36821734 37091336 P1

5 36731578 36978408 P2

5 36908654 37108773 P3

Data Slicer for variants

• Whole genome VCF files are unwieldy• They contain all variants in the genome• They contain all genotypes from all individuals studied• Sometimes you just want to analyse a small region and one

population• The Data Slicer allows you to take a slice of a VCF and narrow

down to only individuals and populations of interest

• Data Slicer currently only accesses the 1000 Genomes data• It is only available for human and only on GRCh37

Hands on – Data Slicer

• I want to get a VCF of the region containing the MC1R gene for the British population

• MC1R is found at 16:89978527-89987385 in GRCh37• The three-letter code for the British population in 1000

Genomes is GBR

FTP

• Files of our complete database:• Genomic, cDNA, CDS, ncRNA and protein sequence

(FASTA)

• Annotated sequence (EMBL, GenBank)

• Gene sets (GTF, GFF)

• Whole-genome multiple and gene-based multiple alignments (MAF)

• Variants (VCF, GVF)

• Constrained elements (BED)

• Regulatory features (BED, BigWig)

• RNA-Seq files (BAM, BigWig)

• MySQL database

Access FTP

Your favourite FTP client

FTP downloads pagehttp://www.ensembl.org/info/data/ftp/index.html

FTP siteftp://ftp.ensembl.org/pub/

FTP files are big

• Multiple Mb/Gb

• Lots of time to download/unzip

• Do you really need this data?

• Make sure it’s the right file before you download.

File chameleon for NGS analysis

• Although files on the Ensembl FTP site are in a standard format, different tools define the standards differently (sigh!)

• Your NGS analysis tool might need files that are slightly different to the Ensembl formats

• File chameleon allows you to download files with these adjustments

Hands on – File Chameleon

• I need a GFF3 file of cat for my RNA-seq analysis.• My tool requires:

• UCSC-style chromosome naming like chr1• Only genes shorter than 4 Mb• Transcript IDs in every line

• We will use File Chameleon to download this customised file.

Analyse your own variants with the VEP

• Find out the effects of your own variants on Ensembl genes• Analyse whole genome variant calls• Filter variants to find those that might be interesting

Your own variant dataVariant coordinates 1 881907 881906 -/C +

5 140532 140532 T/C +12 1017956 1017956 T/A +2 946507 946507 G/C +14 19584687 19584687 C/T -

HGVS notation ENST00000285667.3:c.1047_1048insC5:g.140532T>CNM_153681.2:c.7C>TENSP00000439902.1:p.Ala2233AspNP_000050.2:p.Ile2285Val

VCF #CHROM POS ID REF ALT20 14370 rs6054257 G A20 17330 . T A20 1110696 rs6040355 A G,T20 1230237 . T .

Variant IDs rs41293501COSM327779rs146120136FANCD1:c.475G>Ars373400041

Variation types

1) Small scale in one or few nucleotides of a gene

• Small insertions and deletions (DIPs or indels)

• Single nucleotide polymorphism (SNP)

A G A C T T G A C C T G T C T - A A C T G G AT G A C T T G A C - T G T C T G A A C G G G A

2) Large scale in chromosomal structure (structural variation)

• Copy number variations (CNV)

• Large deletions/duplications, insertions, translocations

deletion duplication insertion translocation

Variation consequences

ATG AAAAAAA

Regulatory

3’ UTRIntronic

CODINGNon-synonymous

CODINGSynonymous

Splice site5’ Upstream 5’ UTR 3’ Downstream

http://www.ensembl.org/info/docs/variation/predicted_data.html

Consequence terms

Predicting missense effects – SIFT and PolyPhen

SIFT and PolyPhen score changes in amino acid sequence based on:

• How well conserved the protein is

• The chemical change in the amino acid• 3D structure and domains (PolyPhen only)

• SIFT and PolyPhen are predictions, not facts• A prediction will never be as good as experimental validation

SIFT PolyPhen

1

0

0.05Deleterious

Tolerated

1

0

0.1Probably damaging

Benign

0.2Possibly damaging

Use the VEP

http://www.ensembl.org/info/docs/tools/vep/index.html

Species that work with the VEP

+ everything in Plants, Fungi, Metazoa, Protists and Bacteria

?

Set up a cache

- Speed up your VEP script with an offline cache.- Use prebuilt caches for Ensembl species.- Or make your own from GTF and FASTA files -

even for genomes not in Ensembl.

http://www.ensembl.org/info/docs/tools/vep/script/vep_cache.html

✓

VEP plugins

• Plugins add extra functionality to the VEP• They may extend, filter or manipulate the output of the VEP.• Plugins may make use of external data or code.• Available on the web tool and with the script.

Hands on

• We’re going to look at a set of four variants to find out what genes they hit and what effect they have on them.

9 128328461 128328461 A/- + var1

9 128322349 128322349 C/A + var2

9 128323079 128323079 C/G + var3

9 128322917 128322917 G/A + var4

Questions?

• We’ve muted all the mics• Ask questions in the Chat box in

the webinar interface• I will check the Chat interface• There’s no threading so please

respond with @name

Host an Ensembl course

Browser course

½-2 day course on the Ensembl browser, aimed at wet-lab scientists.

One trainer.

REST API course

1-2 day course on the Ensembl Perl API, aimed at bioinformaticians.

1-2 trainers.

http://training.ensembl.org/

We can teach an Ensembl course at your institute for free (except trainers’ expenses).

Email us: helpdesk@ensembl.org

Help and documentationCourse online http://www.ebi.ac.uk/training/online/subjects/11

Tutorials www.ensembl.org/info/website/tutorials

Flash animations

www.youtube.com/user/EnsemblHelpdesk

http://u.youku.com/Ensemblhelpdesk

Email us helpdesk@ensembl.org

Ensembl public mailing lists dev@ensembl.org, announce@ensembl.org

Follow us

www.facebook.com/Ensembl.org

@Ensembl

www.ensembl.info

Publications

Aken, B. et al

Ensembl 2017

Nucleic Acids Research

http://europepmc.org/articles/PMC5210575

Xosé M. Fernández-Suárez and Michael K. SchusterUsing the Ensembl Genome Server to Browse Genomic Sequence Data.Current Protocols in Bioinformatics 1.15.1-1.15.48 (2010)www.ncbi.nlm.nih.gov/pubmed/20521244

Giulietta M Spudich and Xosé M Fernández-SuárezTouring Ensembl: A practical guide to genome browsingBMC Genomics 11:295 (2010)www.biomedcentral.com/1471-2164/11/295

http://www.ensembl.org/info/about/publications.html

Ensembl AcknowledgementsThe Entire Ensembl Team

Funding

Co-funded by the European Union