mapping mutations from zebrafish mutagenic screens...

MAPPING MUTATIONS FROM ZEBRAFISH MUTAGENIC SCREENS USING WHOLE

GENOME SEQUENCING

Greg Baillie | IMB Sequencing Facility | Winter School | 8 July 2015

CARDIOVASCULAR AND LYMPHATIC SYSTEMS

circulatory system. Art. Britannica Online for Kids. Web. 1 July 2015. <http://kids.britannica.com/elementary/art-171939>.

Herbert and Stainier, Nat. Rev. Mol. Cell Biol. (2011)

CARDIOVASCULAR SYSTEMRoles• Transport oxygen and nutrients to tissues, remove

carbon dioxide and waste from tissues• Immune cell circulation and surveillance• Regulation of body temperature, pH, water content

Disorders• Congenital heart defects• Cardiomyopathies• Cardiovascular disease

Known genes• bone

morphogenetic protein (BMP), Notch, WNT, sonic hedghog (SHH)

• Heart of glass (HEG), titin (TTN)


Xin et al., Nat. Rev. Mol. Cell Biol. (2013)

LYMPHATIC SYSTEM


Roles• Removal of interstitial fluids from tissues• Absorbs fats from digestive system• Transports immune cells to/from lymph nodes

Disorders• Lymphadenopathy• Lymphedema• Cancer

Known genes• LYVE1, VEGFC,

SOX18, CCBE1, etc

Stacker et al., Nat. Rev. Cancer (2014)

ZEBRAFISH (Danio rerio)

• Phylum: Chordata• Subphylum: Vertebrata• Superclass: Osteichthyes• Class: Actinopterygii• Order: Cypriniformes• Family: Cyprinidae• Genus: Danio• Species: D. rerio

"Fish evolution" by Epipelagic - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commonshttp://commons.wikimedia.org/wiki/File:Fish_evolution.png#/media/File:Fish_evolution.png

ZEBRAFISH MODEL

• Easier to house and care for than rodents

• Lots of offspring• Embryos develop

outside body, optically transparent

• Genome sequenced• Genetic similarity to

humans• Genetic manipulation

technologies

www.nichd.nih.gov

200-300 per pairing (cf. 5-10 for mouse)

http://www.dailyemerald.com/

www.nc3rs.org.uk

ZEBRAFISH MODEL






technologies Karlstrom and Kane, Development, 1996

http://www.imb.uq.edu.au/ben-hogan

www.nc3rs.org.uk

ZEBRAFISH MODEL






technologies Genome Reference ConsortiumEnsembl

Zebrafish HumanChromosomes 1-25 1-22, X, YTotal bases 1,412,464,843

bp3,234,834,689

bpScaffolds 4,559

(3,452 placed, 1,107 unplaced)249

(190 placed, 59 unplaced)

Scaffold N50 1,551,602 bp 46,395,641 bpCoding genes 26,241 20,774Non-coding genes

6,097 22,493

www.nc3rs.org.uk

ZEBRAFISH MODEL






technologiesHowe et al., Nature (2013)

www.nc3rs.org.uk

ZEBRAFISH MODEL






technologies

www.nc3rs.org.uk

• Targeted gene alteration– CRISPR, TALEN, Morpholino,

etc– Rapid confirmation of

candidate genes

FORWARD GENETIC SCREEN

• Trying to find genes involved in development or disease• Induce random mutations

– Insertional mutagenesis (eg. transposons)– Radiation-induced mutagenesis– Chemical mutagenesis (eg. ENU)

• Screen mutants for phenotype of interest• Map mutation to region of genome• Identify mutated gene

– Sequencing

• Confirm role of gene– KO– Complementation

ENU MUTAGENESIS

• N-Ethyl-N-nitrosourea• Alkylating agent• Preference for A→T base transversions and AT→GC transitions

– Also causes GC→AT transitions• Missense (64%), splice site (26%), nonsense (10%)a

• Bath fish in ENU solution• ~1 mutation every 100,000-150,000 bp

aJustice et al. Hum. Mol. Genet. (1999)

ZEBRAFISH FORWARD GENETIC SCREENlyve1 strain

ENUmutagenesis

a*/A

A/A

A/A

A/A

a*/A

a*/A

A/A

a*/AA/A a*/A

25% a*/a*

Mutantphenotype

F0

F1

F2

HOGAN/SMITH SCREENS AT IMB

• 420 families screened from incrossed F1 fish from heavily mutagenised founder population

• ~50 lymphatic development mutants (Hogan lab)

• ~40 heart development mutants (Smith lab)• Largest zebrafish screen completed in Australia• Now in mapping, gene characterisation, and

confirmation phase– 24 lymph mutants sequenced– 16 heart mutants sequenced– 6 heart/lymph mutants sequenced

ZEBRAFISH FORWARD GENETIC SCREENqWIK strain

a*/A

A/A

A/A

A/A

a*/A

a*/A

A/A

a*/AA/A a*/A

25% a*/a*

Mutantphenotype

F0

F1

F2

TRADITIONALMAPPING

• Microsatellites• Coarse mapping - to

identify chromosome (weeks/months)– Pools of fish

• Fine mapping - to identify region of chromosome (weeks/months)– On individual embryos (100s-

1000s)

• Sequence pools of "mutant" and ”reference" fish (3 days)– ~8 genomes per

NextSeq 500 run• Analyse (1 day)

– Map against reference genome (Zv9) [BWA]

– Call variants [GATK]– Identify mutant region

by homozygositymapping

SEQUENCING-BASEDMAPPING

ZEBRAFISH FORWARD GENETIC SCREENlyve1 strain

a*/A

A/A

A/A

A/A

a*/A

a*/A

A/A

a*/AA/A a*/A

25% a*/a*

Mutantphenotype

a*/A

A/A

A/A

A/A

a*/A

a*/A

A/A

a*/AA/A a*/A

qWIK strain

25% a*/a*

Mutantphenotype

ZEBRAFISH FORWARD GENETIC SCREEN

lyve1 strain

qWIK strain

25% a*/a*

Mutantphenotype

40X

13X 20X

8-20X

ANALYSIS PIPELINElane 1fastqs

Map [BWA MEM]Sort [Picard]

sorted.1.bam

danRer7.fa

realign.bam

MarkDuplicates [Picard]Realign [GATK]

lane 2fastqs

lane 3fastqs

lane 4fastqs

MMerge [Picard]




sorted.2.bam

sorted.3.bam

sorted.4.bam

mutant.vcf

Genotype [GATK]

mutant.ref.vcf

Genotype [GATK]

coverage.py

coverage.json

lyve1.bam

qWIK.F.bam

qWIK.M.bam

danRer7.fa

homozygosity.bedgraph


ANALYSIS PIPELINE

SNP Impact [SnpEff]

mutant.snpeff.vcf

homozygosity_calculator.py find_candidate_snps.py

candidates.vcf

Annotate [SnpSift]

candidates.snpeff.vcf


coverage.json

snpeff_to_html.py

Summary.html

mutant.vcfmutant.ref.vcf

fish_conservation.bedgraph

hvertebrate_conservation.bedgrap

hME1.bed

ME3.bed

PyVCF

PyVCFPySam

HOMOZYGOSITY MAPPING

Adapted from Leshchiner et al., Genome Res.2012

mutation

mutant pool

lyve1

qWIK

Homozygous lyve1 region

HOMOZYGOSITY MAPPING

mutation

mutant pool

danRer7 referencegap

window size step size

HOMOZYGOSITY SCORE

(Henke et al)# Mutant hom

# Mutant het ×# qWIK not in mutant

# qWIK in mutant

# mutant hom lyve# lyve informative and qWIK informative

# mutant het lyve/qWIK ×

HOMOZYGOSITY PLOTS

HOMOZYGOSITY PEAKS

Screen Defined Peak

Broad Peak No peak

Heart 8 1 7

Lymph 15 4 5

Heart/Lymph 2 2 2

TRADITIONALMAPPING

• Re-sequence genes in region– PCR, Sanger

sequencing

• Use sequence data– Find candidate

mutations– Estimate mutation

impact

SEQUENCING-BASEDMAPPING

MUTATION DETECTION• Find candidate mutations

– Python– Relaxed

• Mutant covered• At least one of reference samples is covered• Majority allele in mutant not majority in reference samples

– Strict• Mutant is ‘homozygous’• All reference samples covered• Mutant allele absent in all reference samples

• Assess impact– SnpEff (vs Ensembl annotation)

• HIGH, eg. Coding start lost, coding stop gained, splicing, frameshift, etc• MODERATE, eg. Non-synonymous coding change, etc• LOW, eg. Synonymous coding change, etc• MODIFIER, eg. Intron, upstream, downstream

– Conservation in fish, vertebrates

• HTML output

PREMATURE STOP CODON

SPLICE SITE DONOR/ACCEPTOR

MUTATION DETECTIONNON-SYNONYMOUS SUBSTITUTION

MUTATION DETECTIONNON-SYNONYMOUS or NONSENSE?

MUTATION TYPES SUMMARY

Known Unknown CountClear homozygosity peak 33

Single candidate 13 14 27Nonsense 7 8 16

Non-synonymous 1 5 6

Splice donor/acceptor 4 1 4

Intron/upstream/downstream 1 0 1

Multiple candidates 5No candidates 1

No homozygosity peak 13

VALIDATION (mafba)

v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog Ba

VALIDATION (mafba)

Koltowska et al. 2015 (In press)

SUMMARY• Comprehensive screen of genes involved in heart

and lymphatic development ongoing• Whole genome sequencing enables rapid

mapping– Days/weeks (cf. months/years)

• Can also identify causative mutation (sometimes)• Sequencing parental fish decreases per-sample

cost (for large screens)• Combination of mapping techniques (traditional

and sequencing) and new in vivo techniques (eg. TALENs) allows rapid identification of genes involved in vertebrate development

ACKNOWLEDGEMENTS

TAFT LABRyan Taft

Christine EnderKe-Lin Ru

Michael ClarkDarya Vanichkina

Anupma ChoudharyAndrew Calcino

Hyun Jae ‘Josh’ Lee

HOGAN LABBen Hogan

Kaska KoltowskaScott Paterson

Neil BowerChristine Neyt

Anne LagendijkSungmin BaekBaptiste Coxam

Joelle Kartopawiro

SMITH LABKelly SmithSam Capon

Daniela GrassiniJessica De Angelis

QCMG SEQUENCINGDave Miller

Ivon HarliwongSenel Idrisoglu

Suzanne ManningEhsan Nourbakhsh

Craig NourseQCMG IT

John PearsonLynn Fink

Scott WoodDarrin Taylor

Conrad Leonard

QCMGSean Grimmond

Peter Wilson

SIMONS LABCas SimonsJo Crawford

Jason da SilvaDoug Stetner

ISFAngelika Christ

Tim Bruxner

IMB Sequencing Facility

Let our team help you with library preparation and sequencing on Illumina NextSeq 500and MiSeq platforms.

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[email protected]

HOMOZYGOSITY MAPPINGmutation

mutant pool

danRer7 referencegap

window size step size

homozygosity_calculator.pyinput_vcfmutant_idoutput_prefix[gaps_file]parse_vcf_number_of_snps()parse_vcf_physical_distance()

WindowControllerlist windowsint start_indexadd_window()create_windows_for_contig(contig,

contig_coords, window_size=10000, step_size=1000, window_type=None)

update_count_in_windows_for_keys(pos, keys)clear_windows()

SnpWindowstring chromosomeint startint endint segment_startint segment_endint window_sizeint step_sizeCounter countswiggle_start(prev_window=None)wiggle_end(next_window=None)

ContigCoordinatesdict contig_lengthsdict gap_coordsget_contig_lengths_from_vcf(vcf_reader)get_gap_coords_from_bed_file(bed_file)gapped_contig_coords_for_contig(contig)

mapping mutations from zebrafish mutagenic screens...

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