Function Annotation of Animal Genomes (FAANG)

Alan L. Archibald The Roslin Institute and Royal (Dick) School of Veterinary Studies

University of Edinburgh

Genotype - phenotype • Aim

– To predict outcomes • Efficacy of drug • Susceptibility to cancer • Performance of daughters of elite dairy bull • Susceptibility to nematode infections

• Discovery – From phenotype to genotype (gene)

• Prediction – From genotype to phenotype – From sequence to consequence

Phenome

Growth

Feed efficiency

Body composition

Disease resistance

Adapted from Ritchie et al. 2015 Nature Reviews Genetics 16: 85

From sequence to consequence

Value of domesticated animals

• Basic biology • Models for human biology • Agriculture

• Genetic / phenotypic diversity • Genotype to phenotype

Evolution, selection, adaptation • Domesticated animal genomes

– Wide evolutionary range • Shellfish, insects, fish, birds, mammals

• Sequence variation, function, time – Long time scales (millions of years)

• Evolution, speciation – Shorter time scales

• Domestication, signatures of (artificial) selection

Biomedical models

• Pigs, sheep, dogs, chicken,…. • Monogenic inherited diseases (coding sequence) • Genetically modified mice • Failure to recapitulate phenotype • Need better models (e.g. CF pig, HD sheep)

– i.e. understanding of genotype to phenotype

Prediction - success

• Selective breeding • Animal model (phenotypic selection) • Genomic selection (genotypic selection) • Black boxes – lack of understanding of mechanisms

Genomic selection • GS theory developed in 2001 before technology available • First 50K SNP chip (cattle) 2008; 650K in 2010 • GS implemented in all major livestock sectors in developed

world • GS is underpinning faster, more accurate and sustainable

genetic improvement • From SNPs to sequence (via imputation) • Adding knowledge of SNP effects

– Coding/non-coding; known/predicted

Successes – from association to causation

• DGAT1 – dairy cattle, milk yield

• Callipyge – sheep, muscling • MSTN – sheep, muscling • IGF2 – pigs, muscling • Noteworthy

– Regulatory sequences, epigenetics

• One gene at a time: slow, inefficient

Genome-wide • Genome projects

– International collaborative consortia – Major domesticated species – draft(y)/draughty genomes

• Unannotated genome = limited value • State of annotation

– Coding sequences, SNPs – Missing: transcript complexity, regulatory sequences

Status of annotated genomes CHICKEN PIG SHEEP COW HUMAN MOUSE

Assembly Galgal4 Sscrofa10.2 Oar_v3.1 UMD3.1 GRCh38 GRCm38.p2Database version 77.4 77.102 77.31 77.31 77.38 77.38Base Pairs 1,072,544,763 3,024,658,544 2,534,344,180 2,649,685,036 3,381,944,081 3,480,955,279Golden Path Length 1,046,932,099 2,808,525,991 2,619,054,388 2,670,422,299 3,096,649,726 2,730,871,774Genebuild by Ensembl Ensembl Ensembl Ensembl Ensembl EnsemblGenebuild method Full genebuild Full genebuild Mixed strategy build Full genebuild Full genebuild Full genebuildGenebuild last updated Dec-13 Feb-14 Dec-13 Sep-11 Aug-14 Jul-14Coding genes 15,508 21,630 20,921 19,994 20,364 22,592Small non coding genes 1,558 2,989 3,985 3,825 9,673 5,860Long non coding genes 135 14,817 5,385Pseudogenes 42 568 291 797 14,415 7,377Gene transcripts 17,954 30,585 27,099 26,740 196,345 99,934Total genes 17,066 24,754 24,906 23,819 44,854 33,837Ratio transcripts/gene 1.1 1.2 1.1 1.1 4.4 3.0

Reference genome improvement • PacBio long read technology

– Pbjelly • Sheep, cattle (Baylor); Chicken (WashU)

• de novo assembly – Goat, pig, sheep, cattle

• Disruptive technology, multiple genome(s) assemblies – Annotation - “Best in genome” – Graph visualization, but alignment tools not available

Discovering functional sequences • Evolutionary

– Sequence comparison, conservation

– 1000G, G10K,… – Genome sequence sufficient – Conserved, but what is it? – Highly variable ≠ non-

functional

• Functional, biochemical – Assay-by-sequence – *ENCODE, iHEC, Epigenome

roadmap – Expensive – Exploring 4-demensional

space (location + time) – Noise or biologically

meaningful?

ENCODE

Headlines

80.4% participates in at least one biochemical RNA- and/or chromatin-associated event in at least one cell type

95% lies within 8 kilobases (kb) of a DNA–protein interaction (as assayed by bound ChIP-seq motifs or DNase I footprints)

99% is within 1.7 kb of at least one of the biochemical events measured by ENCODE

Summary of the coverage of the human genome by ENCODE data.

Manolis Kellis et al. PNAS 2014;111:6131-6138

©2014 by National Academy of Sciences

Headlines

It is possible to correlate quantitatively RNA sequence production and processing with both chromatin marks and transcription factor binding at promoters

indicating that promoter functionality can explain most of the variation in RNA expression.

Headlines

Single nucleotide polymorphisms (SNPs) associated with disease by GWAS are enriched within non-coding functional elements, with a majority residing in or near ENCODE-defined regions that are outside of protein-coding genes.

In many cases, the disease phenotypes can be associated with a specific cell type or transcription factor.

See also Hindorff et al 2009 PNAS 106: 9362 88% of trait associated SNPs are intronic / intergenic

The complementary nature of evolutionary, biochemical, and genetic evidence.

Manolis Kellis et al. PNAS 2014;111:6131-6138

©2014 by National Academy of Sciences

Epigenetic and evolutionary signals in cis-regulatory modules (CRMs) of the HBB complex.

Manolis Kellis et al. PNAS 2014;111:6131-6138

©2014 by National Academy of Sciences

~$150 million

Functional Annotation of ANimal Genomes (FAANG)

An international collaborative programme

• By-product of biology-led research – development, differentiation, responses to perturbation

• Focus on target tissues – musco-skeletal – immune tissues

• Limited assays – DNaseI, FAIREseq, ATAC-seq – histone marks (promoters, enhancers) – Methylation (BS-seq, RRBS) – RNAseq (stranded), CAGE

Functional Annotation of ANimal Genomes (FAANG)

Virtuous cycle

Experimental data

Improved function annotation

FAANG annotation

pipeline

Re-analyse re-interpret

Transcriptomics discovery, differential expression

• From ESTs via microarrays to RNAseq • Expression atlases

• Pig: microarray (published), RNAseq (in progress) • Sheep: RNAseq (‘pilot’ data used for annotation, in progress) • Chicken: RNAseq (in progress), Avian RNA-Seq Consortium (Burt & Smith

2015) • Buffalo: RNAseq (project funded)

• Transcript complexity – Assembly from short reads challenging – Full length transcript sequences - PacBio

CHICKEN PIG SHEEP COW HUMAN MOUSEAssembly Galgal4 Sscrofa10.2 Oar_v3.1 UMD3.1 GRCh38 GRCm38.p2Database version 77.4 77.102 77.31 77.31 77.38 77.38Base Pairs 1,072,544,763 3,024,658,544 2,534,344,180 2,649,685,036 3,381,944,081 3,480,955,279Golden Path Length 1,046,932,099 2,808,525,991 2,619,054,388 2,670,422,299 3,096,649,726 2,730,871,774Genebuild by Ensembl Ensembl Ensembl Ensembl Ensembl EnsemblGenebuild method Full genebuild Full genebuild Mixed strategy build Full genebuild Full genebuild Full genebuildGenebuild last updated Dec-13 Feb-14 Dec-13 Sep-11 Aug-14 Jul-14Coding genes 15,508 21,630 20,921 19,994 20,364 22,592Small non coding genes 1,558 2,989 3,985 3,825 9,673 5,860Long non coding genes 135 14,817 5,385Pseudogenes 42 568 291 797 14,415 7,377Gene transcripts 17,954 30,585 27,099 26,740 196,345 99,934Total genes 17,066 24,754 24,906 23,819 44,854 33,837Ratio transcripts/gene 1.1 1.2 1.1 1.1 4.4 3.0

A novel gene model: complete with putative UTRs and alternate transcripts as identified by sequencing full length cDNAs derived from chicken brain using PacBio long read technology.

Sheep gene atlas Texel (ram, ewe, ewe lamb, 16d embryo) 50+ tissues per animal, whole embryo

- Samples acquired, RNA prepared Illumina paired ends (2 x 150 bp)

- > 1 Tb stranded RNAseq data Ensembl RNAseq gene models Funded 3SR, RoslinFoundation

Cerebrum Abomasum Skeletal muscle, biceps Testes, epididymis

Brain stem Rumen Skeletal muscle, longissimus dorsi

Corpus luteum, ovary, ovarian follicles

Tonsil Duodenum Skin (side/back) Uterus, cervix, placenta

Cerebellum Omentum Spleen Mammary gland

Hypothallamus Caecum Mesenteric lymph node

Pituitary gland Colon Precapuslar lymph node

Adrenal gland Rectum Peyer’s patch Whole embryo

Thyroid gland Alveolar macrophages

Bone marrow

Sheep RNAseq gene models & expression profiles

Scale warning

Functional annotation of the sheep genome • BBSRC funded 2013 – 2016

– David Hume, Alan Archibald, Mick Watson, Kim Summers, Bruce Whitelaw; Emily Clark, Iseabail Farquhar

• RNA-seq expression atlas – 2,500 tissue/cells samples (5 aliquots each) – 3 mature males, 3 mature females, embryo time course (+pregnant

ewes), lamb time course – 200 in prep for RNA-seq

• CAGE analysis for TSS and enhancers • Genome editing tests for functionality

CAGE – finding promoters and enhancers

Pig alveolar macrophages: CSF1 TSS (DAH gene)

CAGE – finding promoters and enhancers

Chicken CAGE tags from a range of tissues, visualised and quantified around the ACTA1 locus in the ZENBU browser

Adipose, muscle methylomes

• 180 samples; what is an appropriate summarising strategy? • MeDIP-seq lacks precision / resolution

H3K27me3 in sheep muscle

• By-product of biology-led research – development, differentiation, responses to perturbation

• Focus on target tissues – musco-skeletal – immune tissues

• Limited assays – DNaseI, FAIREseq, ATAC-Seq – histone marks (promoters, enhancers) – methylation – RNAseq (stranded), CAGE

Functional Annotation of ANimal Genomes (FAANG)

Data management, sharing and publication • Data hubs model (ENCODE) • Toronto Statement of pre-publication data sharing

Data Flow in FAANG: The data generated by the FAANG consortium will use standard lab protocols and be submitted to EMBL-EBI archives and their peers. The DCC will ensure the data are available to the DAG and collect the analysis results back so they can be released to the wider community and viewable within the main Genome browsers.

Screen shot of pig H3K4me3 and H3K27ac ChIP-Seq data around the POUF5F1 (OCT4) locus delivered from the Roslin Track Hub and visualised in the Ensembl genome browser.

Published 25 March 2015

Functional Annotation of ANimal Genomes

Core assays Transcription Stranded RNA-seq, total (ribo-depleted), poly A+, small RNAs Chromatin accessibility and architecture ATAC-seq CCTF (ChiP-seq) (insulator) Histone modification marks (ChIP-seq) H3K4me3 – promoters active genes, TSS H3K27me3 – genes silenced by regional modification H3K27ac – active regulatory elements (active vs inactive P & E) H3K4me1 – enhancers and other distal elements

Functional Annotation of ANimal Genomes

Additional assays Methylation Whole genome or Reduced Representation Bisulfite Sequencing Transcription Factor Binding Sites (TFBS) ChiP-seq Genome conformation Chromosme Conformation Capture (CCC) HiC

The FAANG Consortium @faangomics

Funding • USDA-NIFA – Huaijun Zhou, UCDavis • INRA – FrAgENCODE –Elisabetta Giuffra, INRA Jouy-en-Josas • ERC – Martien Groenen, Wageningen • PENCODE – Lusheng Huang, China

• BBSRC Ensembl for farmed animals (2012-15; 2015-19) • BBSRC “Establishing the infrastructure for functional

annotation of farmed animal genomes” • BBSRC “Reading and interpreting the code hidden in farm

animal genomes and epigenomes”

“Reading and interpreting the code hidden in farm animal genomes and epigenomes”

• The Roslin Institute – A Archibald, D Burt, D Hume, D Vernimmen, M Watson

• European Bioinformatics Institute – L Clarke, P Flicek

• The Genome Analysis Centre – M Caccamo, M Clark, F di Palma, D Swarbreck

• submitted 8th January 2015 • Reviews received 24 March 2015

Strategic LoLa application

“Reading and interpreting the code hidden in farm animal genomes and epigenomes”

• Chicken, cattle, pigs, sheep – adult – developmental stages

• Data Coordination Centre

• RNA-seq – PacBio, Illumina (short, long)

• CAGE • ATAC-seq • RRBS • ChIP-seq

– H3K4me3, H3K27me3, H3K27ac, H3K4me1, CCTF, TF

WG

5 training

WG

4 dissemination, com

ms.

WG1 Biological resources

WG3 Data standards, analysis, sharing

Stakeholders incl. researchers, funders, end users, public

WG2 Expt. standards

FAANG-Europe COST Action

International FAANG initiative

Submitted 24 March 2015

• SNP in repressor binding site

• Imprinted • Ideal muscling

mutation • Effective exploitation • Missing from

Sscrofa10.2 • FAANG unlikely to

have discovered?

Sequencing Bioinformatics

Next-generation sequencing • 6x Illumina HiSeq 2500 • 3x Illumina MiSeq Sanger sequencing • ABI 3730

Sequencing • De novo assembly, SNP discovery,

metagenomics, …. Functional genomics • RNA-seq, ChIP-seq, microarray

analyses, … Interpretation • Pathway, network analyses,.. Kit • 232 Intel Xeon 64-bit cores, 6Gb

RAM/core, 264TB high performance storage

Microarrays Genotyping

Technologies • Affymetrix • Agilent • Illumina

Applications • Gene expression • CGH

High density / custom arrays Illumina • iScan, Infinium • BeadXPress, GoldenGate • BeadChip Affymetrix • GeneTitan • Axiom

genomics.ed.ac.uk

genomics.ed.ac.uk

Jan 12, 2015 University of Edinburgh and University of Glasgow and Illumina form The Scottish Genomes Partnership

Scottish Genomes Partnership announce they will install 15 HiSeqX machines

Currently, limited to use for sequencing human samples