Download 2610//16 1 Allele-specific expression, ASE [1] Defini8on of allele

2610//16
SciLifeLabRNA-seqcourse
Allele-specificexpression,ASE
OlofEmanuelsson
KTHRoyalInsBtuteofTechnology
[email protected]
2016-10-27,11:00-12:00Navet(E10),BMC,Uppsala
[1]Defini=onofallele-specific
expression(ASE)
Addinganotherlayertotranscriptomecomplexity...
♀
♂
Adopted from Unneberg, 2010
...andeachgeneispresentontwochromosomes.
=>ithastwoalleles
ASE:allele-specificexpression
Outline
1.  Defini=onofASE
2.  Detec=ngASE(introductorycase)
3.  Applica=onsandprevalenceofASE
4.ImportantASEconsidera=ons
(a)Variantcalling
(b)MappingbiasASEtools
(c)Manyvariantsinagene
5.ASEtools
6.  GeneiASE–atooltodetectgeneswithASEfromRNA-
seqdata
Addinganotherlayertotranscriptomecomplexity...
Adopted from Unneberg, 2010
Onegenecanproducemanydifferenttranscripts...
Allele,defini=on
Analleleisthevariantformofagivengene(orlocus).
SomeBmes,differentallelescanresultindifferent
observablephenotypictraits,suchasdifferentpigmentaBon.
/…/
Ifbothallelesatagene(orlocus)onthehomologous
chromosomesarethesame,theyandtheorganismare
homozygouswithrespecttothatgene(orlocus).Ifthe
allelesaredifferent,theyandtheorganismare
heterozygouswithrespecttothatgene(orlocus).
hXps://en.wikipedia.org/wiki/Allele
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Allele-specificexpression,defini=on
AnimbalanceintranscripBonbetweenthematernaland
paternalallelesatalocus.
•  I.e.,adevia=onfromtheexpected50/50ra=oof
transcripBonfromthetwoallelesofadiploidorganism.
•  Canbeassessedwithinasingleindividual
(Presentalsowhenploidy>2,e.g.,plants)
Othereventsmayalsobe“allele-specific”,e.g.
•  transcripBonfactorbinding
•  DNAbackbonemethylaBon
•  X-chromosomeinacBvaBoninfemalemammals
Detec=ngallele-specificexpression
Wetlabtechnologies:
•  microarrays(ifdesignedproperly)
•  qRT-PCR+TaqMan
•  pyrosequencing
•  RNA-seq
N.B.:asthesearesequence-basedtheywillnotprovideany
informaBoninthecaseofahomozygousallele,althoughit
maysBllbeexpressedpredominantlyfromonlyoneofthe
chromosomes.
eQTL–expressionquan=ta=vetraitloci
Anotherapproach!
Requiresmanysubjects
[2]Detec=ngASE
Detec=ngallele-specificexpressionusingRNA-seqdata
•  RNA-seqreadsprovidethesequenceofatranscript
•  ...whichenablesthedeterminaBonoftheallelicorigin
ofthereadsoverlappingwiththeSNV
Diploidgenome
SNV
Allele-specificgene
expression(mRNAs)
U
U
Allele-specificexpression,defini=on
genomicDNA->transcript(e.g.mRNA)
Allele-specificgene
Diploidgenome
expression(mRNAs)
U
U
SNV
•  SNV=singlenucleoBdevariant
•  ThegenomicSNVisreflectedinthetranscribedRNA(Tis
UinRNA).
RNA-seqreads
T
T
C
Detec=ngallele-specificexpressionusingRNA-seqdata
Generaloutline:
1.MaptheRNA-seqreads
2.Countthereadsthatmaptoeitherallele
3.Calculateeffectsizeandp-value
C
C
C
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Detec=ngallele-specificexpressionusingRNA-seqdata
1.MaptheRNA-seqreads
Detec=ngallele-specificexpressionusingRNA-seqdata
1.MaptheRNA-seqreads
Detec=ngallele-specificexpressionusingRNA-seqdata
2.Countthereads
Detec=ngallele-specificexpressionusingRNA-seqdata
3.Calculateeffectsizeandp-value
Paternalallele(a) Maternalallele(A)
…AGTCTTCCAATTAGC…
…AGTCTTCTAATTAGC…
Reads–10xcoverageofthelocus
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCCAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCCAATTAGC…
…AGTCTTCCAATTAGC…
Paternalallele(a) Maternalallele(A)
…AGTCTTCCAATTAGC…
…AGTCTTCTAATTAGC…
Mappedreads
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCCAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCCAATTAGC…
…AGTCTTCCAATTAGC…
Paternalallele(a) Maternalallele(A)
3x …AGTCTTCCAATTAGC…
7x …AGTCTTCTAATTAGC…
3readsmappedtopaternalallele
7readsmappedtomaternalallele
Intotal10readsmappedtothelocus
Effectsize:(otherdefiniBonspossible)
ASEeffect=calt/(calt+cref)–0.5
i.e.,thefracBonofcountsmappedtoalternaBvealleleminus0.5=>
ŸifnoASEthenASEeffect=0
ŸrangeofASEeffectis[-0.5,0.5]
P-value:Usebinomialwithp=0.5(assuming50/50transcripBon)
Ourexamplefrompreviousslide:
Effectsize=ASEeffect=calt/(calt+cref)–0.5=3/(3+7)-0.5=–0.2
P-value:binomialtestfordeviaBonfrom50/50distribuBonbetween
alleles(inR):
> pbinom(3, size=10, prob=0.5)
[1] 0.171875
⇒ NotsignificantinthisparBcularexample
⇒ Ifcoveragewas30x(9+21reads)insteadof10x(3+7),thenp-value<0.03
eQTLvs.ASE
eQTL
ASE
• Sufficientpowerwithasingle
• Inter-individualdifferencesinexpression
individual
• Modesteffects
• IdenBcalcellularenvironmentfor
• LargenumberofSNP-genecombinaBons
thetwochromosomes
• Manysamplesneeded
• NoassociaBontoregulatoryregion
• Mayusemicroarraysforgeneexpression
• MustuseRNA-seqforgene
• Genotypingrequired
expression
[3]Applica=onsandprevalenceofASE
10individualsgenotyped
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Applica=onofASE
Applica=onofASE
Findproteinvariants
Findcis-regulatoryvariant
Allele-specificgene
expression(mRNAs)
cis-regulatory
variant
U
U
SNV
PossibletodetectifyoualsohaveinformaBonaboutnon-transcribed
variants(e.g.,fromwhole-genomeDNAsequencingorSNP-array).
Diploidgenome
Allele-specificgene
expression(mRNAs)
Differentproteins
U
U
SNV
Toinferachangedprotein,theSNVmustbe
•  incodingregion
•  non-synonymous
Applica=onofASE
Normalvs.tumorexpression
Possibletodetectifyouhaveexpressionmeasuredfrombothnormaland
tumorBssue(inthesameindividual).
PrevalenceofASE
GeneswithsignificantASE(%ofgeneswithheterozygousvariant).
ImportantASEconsidera=ons
[4]ImportantASEconsidera=ons
(a)  Variantdetec=on
(b)Mappingbias
(c)Manyvariantsinagene
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Variantdetec=on
[4]ImportantASEconsidera=ons:
(a)Variantdetec=on
Variant=aposiBoninthegenomethatisdifferentfromanothergenome.
•  Homozygousvariant:thetwoallelesareidenBcaltoeachother
•  Heterozygousvariant:thetwoallelesaredifferent
•  “Ref.”=thealleleisthesameasforthereferencegenome
•  “Alt.”=alternate=thealleleisdifferentfromthereferencegenome
•  SNVisonetypeofvariant,othersincludeinserBon,deleBon,...
Variantdetec=on=detecBngwhatvariantsarepresentinasample:
1.  Variantcalling–anyposiBonwithevidenceofanalternaBvebase
2.  VariantprioriBzaBon–definereliablevariantswithhighconfidence
TypicallyperformedbasedongenomicDNAdata,from
•  Microarrays(e.g.IlluminaOmni2.5M)
•  Sequencing(e.g.whole-genomere-sequencingorexomesequencing)
Variantdetec=onfromsequencingdata
Startbymapthereads.
Paternalallele(a) Maternalallele(A)
…AGTCTTCCAATTAGC…
…AGTCTTCTAATTAGC…
Reads–10xcoverageofthelocus
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCCAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCCAATTAGC…
…AGTCTTCCAATTAGC…
Variantdetec=onfromsequencingdata
Thisiswhatweactuallyhave:
Paternalallele(a)
Maternalallele(A)
Referencesequence
…AGTCTTCCAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
Mappedreads
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCCAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCCAATTAGC…
…AGTCTTCCAATTAGC…
=>needtodetectthevariantposiBonsinthereferencesequence
Variantdetec=onfromsequencingdata
OK,pieceofcake?
Paternalallele(a) Maternalallele(A)
…AGTCTTCCAATTAGC…
…AGTCTTCTAATTAGC…
Mappedreads
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCCAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCTAATTAGC…
…AGTCTTCCAATTAGC…
…AGTCTTCCAATTAGC…
Variantdetec=onfromsequencingdata
Standard:GATK(DePristoetal.,2011)orSamtools–worksonanymapped
sequencingdata.
GATKscorestheSNVsbytakingintoaccountanumberofcharacterisBcs,
including:
•  Sequencingdepth(coverage)
•  Mappingquality
•  PosiBonbias(basequality)
SpecificRNA-seqbasedtools:
•  Colib’read–LeBrasetal.,2016
•  RVboost–Wangetal.,2014
•  ACCUSA2–PiechoXaetal.,2013
GATKthemostwidelyused,evenforRNA-seq.
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Variantdetec=on–whichvariantstouse(priori=za=on)? VCFisatextfileformat(“flattext”).ExampleVCFoutputfromGATK:
VariantsfromRNA-seq
•  Whatsequencingdepth?
influencesthepower,seeè
•  Heterozygous
•  Othercriteria
Filteringofknownvariants
•  KeeponlyvariantsindbSNP?
##fileformat=VCFv4.1
...
#CHROM POS
ID
REF ALT QUAL FILTER INFO FORMAT SAMPLE1_NA12878 [SAMPLE1_BLALBA] ...
1 873762 . T C 5231.78 PASS [ANNOTATIONS] GT:AD:DP:GQ:PL 0/1:173,141:282:99:255,0,255 ...
1 877664 rs3828047 A G 3931.66 PASS [ANNOTATIONS] GT:AD:DP:GQ:PL 1/1:0,105:94:99:255,255,0...
1 899282 rs28548431 C T 71.77 PASS [ANNOTATIONS] GT:AD:DP:GQ:PL 0/1:1,3:4:26:103,0,26 ...
1 974165 rs9442391 T C 29.84 LowQual [ANNOTATIONS] GT:AD:DP:GQ:PL 0/1:14,4:14:61:61,0,255...
...
GT:thegenotypeofthissampleatthissite(0/0,0/1,1/1,1/2,...).0=ref.,1=alt.
AD:alleledepths,i.e.,thenumberofreadsthatsupporteachofthereported
alleles
GQ:qualityofassignedgenotype(max=99)
FullspecificaBonofVCFfileformat:hXp://samtools.github.io/hts-specs/
AllelicraBo
Variantdetec=on–VCF,VariantCallFormat
:40
:67
:30
:20
50
Sequencingdepth
Mappingbias
[4]ImportantASEconsidera=ons:
(b)Mappingbias
Mappingbias–exampleinrealdata
Heterozygousvariants(alt/ref)mapped
toreferencegenome.
X-axis,alternateallelefracBon[0,1]
Y-axis,density
(Datafrom16RNA-seqexperiments;
VariantsdetectedwithRNA-seqdata
orSNParray).
Referencegenomevariants(“ref.”)haveanadvantageinthemapping.
Maternalallele:…ATCGAATGAAGCTCATTGGATCAGAT… (ref.)
Paternalallele: …ATCGAATGAAGCTTATTGGATCAGAT… (alt.)
Reference: …ATCGAATGAAGCTCATTGGATCAGAT…
Mappingofreads
Readfrommaternalallele: AGCTCATT
::::::::
Reference: ATCGAATGAAGCTCATTGGATCAGAT
:::: :::
Readfrompaternalallele: AGCTTATT
Thepaternalallelereadwillmapwithalowermappingquality.
IncaseofsequencingerrororpoorbasequalityatanotherposiBon,this
mightpushthemappingqualityofthepaternalallelereadbelowthe
threshold,andthereadwillbediscarded.
Mappingbias–waystogetarounditinASEdetec=on
Maskingvariants({A,C,G,T}=>N)
YoulooseinformaBon.
ConstructallpossibleversionsofthegenomefromexisBngvariants
CansoongenerateaprohibiBveamountofgenomeversions.
Mapreadstodiploidgenome(ortranscriptome)
Requiresthatyoueitherhaveorconstructthediploidgenome(or
transcriptome)oftheindividual.
Modfiythebinomialprobabilityptoreflectthemappingbias.
RequiressimulaBontoproperlymodifyp.
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Mappingbias–waystogetarounditinASEdetec=on
Maskingvariants({A,C,G,T}=>N)
YoulooseinformaBon.
ConstructallpossibleversionsofthegenomefromexisBngvariants
CansoongenerateaprohibiBveamountofgenomeversions.
Mapreadstodiploidgenome(ortranscriptome)
Requiresthatyoueitherhaveorconstructthediploidgenome(or
transcriptome)oftheindividual.E.g.,Turroetal.(2011)(transcriptome).
Modfiythebinomialprobabilityptoreflectthemappingbias.
RequiressimulaBontoproperlymodifyp.E.g.,Montgomeryetal.(2010).
[4]ImportantASEconsidera=ons:
(c)Manyvariantsinagene
Manyvariantsinagene
Manyvariantsinagene
Morethanonevariantwithinageneiscommon:
Allele-specificgene
Diploidgenome
expression(mRNAs)
A U
A U
A
2different“haploisoforms”
G C
G
G C
SNV_1SNV_2
G C
RNA-seqcontainsinformaBonthattherearetwoheterozygousSNVs.
Allele-specificgene
Diploidgenome
RNA-seqreads
expression(mRNAs)
A
T
A U
T
A U
A
A
G C
G
G
G C
C
G
SNV_1SNV_2
G C
C
G C
G
G
G C
C
G
C
VariantsdetectedfromRNA-seqreads:
•  A/Gatonelocus
•  T/Catonelocus
Manyvariantsinagene
Manyvariantsinagene–phasing
But:RNA-seqdoesnotnecessarilycapturetherelaBonbetweenthetwo
SNVs.
Allele-specificgene
Diploidgenome
RNA-seqreads
expression(mRNAs)
G
C
A U
T
A U
A
C
G
C
G C
G
G
G C
C
SNV_1SNV_2
G C
A
G
T
G C
G
A
G C
PossiblecombinaBons(haplotypes)fromRNA-seqreads:
•  A+TandG+C
•  A+CandG+T
Phasing=decidingwhichallelesthatareonthesamechromosomal
homologue
Diploidgenome
RNA-seqreads
A U
UC
G A
A
T
G
C
G
C
SNV_1SNV_2
G
C
G
A
G
A
T
G
A
PossiblecombinaBons(haplotypes)fromRNA-seqreads:
AT
GC
•  A+TandG+C:and
(2+2different
AC
GT
•  A+CandG+T:and
“haploisoforms”)
ButcanourRNA-seqreadsprovidethephase?
G
G
C
C
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PhasingisusefulbutnotnecessarytodetectASE
PhasinginformaBontypicallyachievedbysequencingthegenomesofthe
parentsofthesubject.Directhaplotypesequencingalsopossible.
Ifyoudon’tknowthephase(andformostRNA-seqdatasets,youdon’t):
•  Trytoinferitfrom
(a)yourRNA-seqdata–possible,buttypicallyonlyparBalphasing
(b)exisBngpopulaBondata(LD)–notapplicableonnewvariants
•  DisregardfromitandcalculateASEanyway
Phasing
•  reducesmappingbias
•  enablesthedetecBonofhaploisoformexpression(isoformsrepresenBng
thetwohomologouschromosomes)
•  butisnotnecessarytodetectASEingeneswith>1SNV
ASEtools
[5]ASEtools
ASEtools–whereonlyRNA-seqdatafromasingle
individualisrequired.
AlistoftoolsthatcandetectASE,givenspecifiedinputdata:
•  cisASE–pairedgenomic+transcriptomicdata,Liuetal.,2016
•  MutRSeq–nonsynonomousSNVsfromRNA-seqdata,Fuetal.,2016
•  GeneiASE–unphasedRNA-seqdata,Edsgärdetal.,2016
•  ASE-TIGAR–parentaldatarequired,bayesian,Nariaietal.,2016
•  ASEQ–pairedgenomic+transcriptomicdata,Romaneletal.,2015
•  MBASED–phasedorunphasedRNA-seqdata,Maybaetal.,2015
•  Allim–parentaldatarequired,Pandeyetal.,2013
•  MMSEQ–aXemptshaploisoformidenBficaBon,Turroetal.,2011
•  (Skelly)–requiresphaseddata,Skellyetal.,2011
•  AlleleSeq–requiresgenomicsequence,Rozowskyetal.,2011
•  (AlleleDB–databaseforASEetc.of1000genomes,Chenetal.,2016)
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
• 
GeneiASE
[6]GeneiASE
cisASE–pairedgenomic+transcriptomicdata,Liuetal.,2016
MutRSeq–nonsynonomousSNVsfromRNA-seqdata,Fuetal.,2016
GeneiASE–unphasedRNA-seqdata,Edsgärdetal.,2016
ASE-TIGAR–parentaldatarequired,bayesian,Nariaietal.,2016
ASEQ–pairedgenomic+transcriptomicdata,Romaneletal.,2015
MBASED–phasedorunphasedRNA-seqdata,Maybaetal.,2015
Allim–parentaldatarequired,Pandeyetal.,2013
MMSEQ–aXemptshaploisoformidenBficaBon,Turroetal.,2011
(Skelly)–requiresphaseddata,Skellyetal.,2011
AlleleSeq–requiresgenomicsequence,Rozowskyetal.,2011
(AlleleDB–databaseforASEetc.of1000genomes,Chenetal.,2016)
GeneiASEdetectsgeneswithsignificantASE,insingleindividualsandbased
onlyonRNA-seqdata.HaplotypeinformaBon(phasing)isnotneeded.
Datarequired:
•  RNA-seqdata
Pre-processingrequired:
•  Mappingandqualitycontrolofreads
•  VariantdetecBon(e.g.,GATK)
•  Filtervariantsifdesired
•  Allelecountsforvariantsextractedintocustominputtextfile
Availability:
•  Edsgärdetal.,Scien9ficReports6:21134,2016
•  hXps://github.com/edsgard/geneiase(GNUGPL3license)
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GeneiASE
Thesitua9on:
Gene i SNV_1 SNV_2 SNV_3 SNV...
•  unphaseddata
Ref
60
20
70
...
•  non-uniformeffectwithingene
Alt
40
80
30
...
•  technicalvariability
TheGeneiASEsolu9on:
1.  Foreachgene,loopoverallitsSNVsandtheir2x1matrixofreadcounts
2.  CalculateateststaBsBc(sij)foreachSNV,basedonreadcounts
3.  CombinetheteststaBsBcsfortheSNVswithinagene=>teststaBsBcfor
enBregene(gi)asdf
4.  ResamplefromparametricnullSNVmodel(esBmatedfromDNAdata)105
Bmes,calculatetheresulBngdistribuBonofgeneteststaBsBc(g0i).
5.  Comparegitog0iandcalculateap-valueforgenei.
GeneiASE–nullmodel,andgene-wisep-valuecalcula=on
0.EsBmateSNVnullmodelparameters
DNAbasedesBmateofthetechnicalvariability
GeneiASE–calculateSNP-basedgene-wiseteststa=s=c
1.CountreadsforeachSNVinagene;add
pseudocountsifrequired
2.CalculateSNVteststaBsBcsijbasedon
absolutevalueofeffectsize,eff.
Absolutevalueofeffectsize=>Undirectedeffect
3.CalculategeneteststaBsBcgiusing
Stouffer-Liptakmethod;kisnumberof
SNVsingenei
RunningGeneiASE–input
Sta=cASE
• geneiase-cvtl-icvtl.test.input.tab
Foreachgene(genei):
1.SampleallelecountsfromnullSNVmodel
(Randomeffectmodel)
2.CalculatekSNVteststaBsBc
k=numberofSNVsingenei
3.CalculategeneteststaBsBc
(Stouffer-Liptak)
Condi=on-dependentASE
• geneiase-icd-iicd.test.input.tab
4.Reiterate1-3NBmes(default:105)
5.Calculatep-valueforgenei
Onelinepergene.
Outputcolumns:
•  feat:FeatureIDasspecifiedintheinputfile(typicallyageneidenBfier)
•  n.vars:Numberofvariantswithinthegene
•  mean.s:Meanofsacrossthevariantswithinthegene
•  median.s:Medianofsacrossthevariantswithinthegene
•  sd.s:StandarddeviaBonofsacrossthevariantswithinthegene
•  cv.s:CoefficientofvariaBonofsacrossthevariantswithinthegene
•  liptak.s:Stouffer-LiptakcombinaBonofs(calledgonpreviousslides)
•  p.nom:Nominalp-value
•  fdr:Benjamini-Hochbergcorrectedp-value
(Reminder:sistheeffectsize-basedteststaBsBcforeachSNVinagene).
RunningGeneiASE–results
Thenumberofgeneswithsignificant(fdr<0.05)ASEasdetectedby
GeneiASEfrom16RNA-seqsamples(primarywhitebloodcells).
Numberofgenes
(with≥2SNVs)
RunningGeneiASE–output
Millionmappedhigh-qualityreads
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Thankyouforyourafen=on
contact:[email protected]
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