MicroArray Evolution:expression to mapping and back again

Justin BorevitzSalk Institutenaturalvariation.org

MicroArray Evolution:expression to mapping and back again

Justin BorevitzSalk Institutenaturalvariation.org

Talk Outline• Natural Variation in Light Response

• Transcriptional profiling– for QTL candidate genes

• Single Feature Polymorphisms (SFPs)– Potential deletions

• Bulk Segregant Mapping– Extreme Array Mapping

• Haplotype analysis

• Expression Revisited

Natural Mutation in PHYA photoreceptorNatural Mutation in PHYA photoreceptor

Quantitative Trait Loci

EPI1 EPI2

differences may be due to expression or hybridization

What is Array Genotyping?

• Affymetrix expression GeneChips contain 202,806 unique 25bp oligo nucleotides.

• 11 features per probset for 21546 genes• New array’s have even more• Genomic DNA is randomly labeled with

biotin, product ~50bp.• 3 independent biological replicates

compared to the reference strain Col

GeneChip

Potential Deletions

Spatial Correction

Spatial Artifacts

Improved reproducibilityNext: Quantile Normalization

False Discovery and Sensitivity

Permuted data

real data

5% FDR

PM only SAM threshold

5% FDR

GeneChip SFPs nonSFPs Cereon marker accuracy 3806 89118 100% Sequence 817 121 696 Sensitivity

Polymorphic 340 117 223 34% Non-polymorphic 477 4 473

False Discovery rate: 3% Test for independence of all factors: Chisq = 177.34, df = 1, p- value = 1.845e- 40

Observed t statistics vsNull (permuted) t statistics

Chip genotyping of a Recombinant Inbred Line

29kb interval

Discovery 6 replicates X $500 12,000 SFPs = $0.25Typing 1 replicate X $500 12,000 SFPs = $0.041

Potential Deletions

>500 potential deletions45 confirmed by Ler sequence

23 (of 114) transposons

Disease Resistance(R) gene clusters

Single R gene deletions

Genes involved in Secondary metabolism

Unknown genes

Potential Deletions Suggest Candidate Genes

deletion of MAF1

FLOWERING1 QTL

Chr1 (bp)

Flowering Time QTL caused by a natural deletion in MAF1

MAF1

Fast Neutron deletions

FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1

Map bibb100 bibb mutant plants100 wt mutant plants

bibb mapping

ChipMapAS1

Bulk segregantMapping usingChip hybridization

bibb maps toChromosome2 near ASYMETRIC LEAVES1

BIBB = ASYMETRIC LEAVES1

Sequenced AS1 coding region from bib-1 …found g -> a change that would introduce a stop codon in the MYB domain

bibb as1-101

MYB

bib-1W49*

as-101Q107*

as1bibb

AS1 (ASYMMETRIC LEAVES1) =MYB closely related toPHANTASTICA located at 64cM

LOD

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eXtreme Array Mapping

Red light QTL RED2 from 100 Kas/ Col RILs

QTL likelihood model using bulk segregant analysis with SFP genotyping

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Composite Interval Mapping

RED2 QTL

Chromosome 2

15 tallest RILs pooled vs15 shortest RILs pooled

RED2 QTL 12cM

Simulation Likelihood

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Chromosome 5 (cM) 50 eXtremeof 500 F2s chr2 QTL 37% varchr5 QTL 13% var

Can detect additive or epistatic QTL but hard to determine which

Fine Mapping with Arrays

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Single Additive Gene1000 F2sSelect recombinantsby PCR 1Mb region

Array Haplotyping

• What about Diversity/selection across the genome?

• A genome wide estimate of population genetics parameters, θw, π, Tajima’D, ρ

• LD decay, Haplotype block size

• Deep population structure?

• Col, Lz, Ler, Bay, Shah, Cvi, Kas, C24,

Est, Kin, Mt, Nd, Sorbo, Van, Ws2

Array Haplotyping

• Hybridize 48 arrays with 15 accessions

• 3 replicates

• Analyze 1/10 Genome 12Mb

• 24,507 features x 14 = 343,098 T-tests

• Spatial Correction, quantile normalization,

SAM like T-tests, permutation null distribution.

Array Haplotyping

Inbred lines

Low effectiverecombinationdue to partialselfing

Extensive LDblocks

Col Ler Cvi Kas Bay Shah Lz Nd

Chr

omos

ome1

~50

0kb

(-4,-3.5] (-3,-2.5] (-2,-1.5] (-1,-0.5] (0,0.5] (1,1.5] (2,2.5] (3,3.5]

T statistic

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Distribution of T-stats

null (permutation)actual

Not Col ColNA NA duplications

32,427Calls

208,729

12,250 SFPs

Diversity

6000000 6200000 6400000 6600000 6800000 7000000

-3-2

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12

3

Chromosome 1 (Mb)

Div

ers

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7000000 7200000 7400000 7600000 7800000 8000000

-3-2

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Chromosome 1 (Mb)

Div

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8000000 8200000 8400000 8600000 8800000 9000000

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Tajima’s D

8000000 8200000 8400000 8600000 8800000 9000000

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jima

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like

25 bp 25 bp

25 bp 25 bp16 bp

25 bp 25 bp8 bp

1st Set

2nd Set

3rd Set

Complete Genome Tiling Chip

Polymorphisms (re-sequencing)Global methylation (Methylome)Comparative Genomics (Brassica)

New Gene DiscoveryImprove AnnotationAlternative SplicingMicro RNAs

9 Whole-Genome Expression Chips2 Splicing Chips2 5’ Mapping Chips

Validate features

Extra Chips:

ChIP – Chip (DNA binding sites)

ChipViewer: Mapping of transcriptional units of ORFeome

From 2000v At1g09750 (MIPS) to the latest AGI At1g09750

2000 v Annotation (MIPS)

The latest AGI Annotation

Expression Analysis Revisited

FLC controls flowering time Difference detected it 3 day old seedlings

Feature level model Gene Expression index that accounts for feature effect and polymorphisms

PAG1 down regulated in Cvi

PLALE GREEN1 knock out has long hypocotyl in red light

Review

• Transcriptional profiling to identify candidate genes and downstream responses that consider polymorphisms

• Single Feature Polymorphisms (SFPs) can be used to identify recombination breakpoints, potential deletions, for eXtreme Array mapping, and haplotyping

NaturalVariation.org

SyngentaHur-Song ChangTong Zhu

SyngentaHur-Song ChangTong Zhu

Salk

Jon WernerTodd MocklerSarah LiljegrenJoanne ChoryDetlef WeigelJoseph Ecker

UC Davis

Julin Maloof

UC San Diego

Charles Berry

Scripps

Sam HazenElizabeth Winzeler

University of Guelph, CanadaDave WolynUniversity of Guelph, CanadaDave Wolyn

Salk

Jon WernerTodd MocklerSarah LiljegrenJoanne ChoryDetlef WeigelJoseph Ecker

UC Davis

Julin Maloof

UC San Diego

Charles Berry

Scripps

Sam HazenElizabeth Winzeler

NaturalVariation.org

Effect of SNP position

340 CandidatePolymorphisms

False negative

True Positive

Fine Mapping LIGHT1

500kb

2nd NorthQTL

(-1,-0.875] (-0.625,-0.5] (-0.125,0] (0.25,0.375] (0.625,0.75]

Tajma's D like statistic

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Future Projects

Design 2nd generation expression array $200

350,000 25mer features, expect 12,000 SFPs (2 accessions)Validated gene models (exon intron)2 features per exon, alternative splicing5’ 3’ Untranslated region for gene family/ polymorphismMicro RNAsValidated “good” hybridization intensities

Haplotype Map –20 Accessions 3 replicates, SFP discovery

estimated 85,000 SFPs 1.4kb resolution

Association Studies –

120 Accessions 1 replicate, Genotyping

Bulk Segregant Mapping –Confirm Associations in specific crosses

Future Projects DNA

True natural variation in gene expression20 Accessions 3 replicates, (polymorphism accounted for)

Cis regulatory variation/ Imprintingreciprocal F1s 3 replicates

Transcriptome QTL Map –100 best VanC Advanced Intercross Lines

How many loci control the variation in gene transcription? Candidate TF and binding sites?

Future Projects RNA

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Pairwise Correlation between and within replicates

LIGHT1 tstat and raw data

LIGHT1 tstat and raw data