a whole genome assembly of rye · cv. julius and barley cv. barke) • wgs and mate-pair libraries...

AWholeGenomeAssemblyofRye(Secalecereale)M.TimothyRabanus-WallaceLeibnizInstituteofPlantGeneticsandCropPlantResearch(IPK)

MTRabanus-Wallace2018


Whyrye?


QTLanalysisforSurvivalAfterWinter(SAW)scoresinrye:

Erathetal.2017

Frost-damageinwinterwheatPhoto:IngridKristjanson(http://cropchatter.com/impact-of-frost-on-winter-wheat-fall-rye/)


RyeSecalecerealeChallenge1)Length(7.9Gbp)

Assemblychallenges…


RyeSecalecerealeChallenge1)Length(7.9Gbp)Challenge2)90+%repetitive



RyeSecalecerealeChallenge1)Length(7.9Gbp)Challenge2)90+%repetitiveChallenge3)Obligateoutcrossing



AssemblyScaffolds


AssemblyScaffolds

MolecularMap


AssemblyScaffolds

MolecularMap

Genome


AssemblyScaffolds

MolecularMap

Genome

Majorryeassemblymilestones:Martis‘13:ARyeProto-Genome(“Zipper”)Bauer‘17:ADraftGenomeIRGSC‘18:AWGSDeNovoGenomeApproachingReferenceQuality…

Martis‘13:ARyeProto-Genome(“Zipper”)•  RNAseq(ExpressedSequenceTags;ESTs)

•  Rawsequenceinformation•  ChromosomeSurveySequencing(CSS)

•  Chromosome-assignedsequenceinformation•  5KSNP-array-basedgeneticmap

•  ESTanchoringbackbone•  Interspeciesgenecolinearity

•  Fine-scaleorderingandgeneidentificationbysequencehomology

MTRabanus-Wallace2018 Martisetal.2013







0

5,000,000

10,000,000

15,000,000

100 10,000 1,000,000 100,000,000Scaffold length bin (bp; bin size = 0.2 log bp)

Sequ

ence

in b

in (b

p)

EST_zipper_2013



•  Chromosomeassignment•  5KSNP-array-basedgeneticmap


•  Fine-scaleESTorderingBauer‘17:ADraftGenome•  WGSandMate-Pair(MP)Libraries

•  Rawsequenceandhierarchicalscaffolding•  CSS

•  Contigandmate-pairreadassignmentpre-scaffolding

•  High-densitySNPmap(iSelectRye600kArray)•  Toanchorscaffolds

•  DArTseqgeneticmap•  Toguidescaffoldinganddetectchimeras

•  Martis’13genomezipper(updated)MTRabanus-Wallace2018

Baueretal.2017

Chr1Rcontigs

Unassignedcontigs

ShortMPreads





•  Fine-scaleESTorderingBauer‘17:ADraftGenome•  WGSandMate-PairLibraries


•  Contigandmate-pairassignmentpre-scaffolding




0

5,000,000

10,000,000

15,000,000


Sequ

ence

in b

in (b

p)

EST_zipper_2013











0

300,000,000

600,000,000

900,000,000


Sequ

ence

in b

in (b

p)

EST_zipper_2013











0

300,000,000

600,000,000

900,000,000


Sequ

ence

in b

in (b

p)

EST_zipper_2013

Bauer_2017


2018:ApproachingReferenceQualityAnNRGeneDeNovoMAGIC3.0assembly(analoguesinwheatcv.Juliusandbarleycv.Barke)•  WGSandmate-pairlibraries

•  Rawdata•  Map-anchoredcontigs(fromBauer‘17)

•  Preliminaryanchoring,chromosomeassignmentandchimeradetection

•  10xChromiummolecule-linkedreads•  Long-rangescaffoldinginformation•  Chimerabreakpointdetection

•  CSS•  Chromosomeassignmentandchimeradetection

…andupcoming…

•  PopSeqhigh-densitygeneticmapping•  Mapanchoringandchimeradetection

•  Chromosome-ConformationCaptureSequence(Hi-C)•  Fine-scaleorderingandorientationfor

pseudomoleculeconstruction

10Xmolecule-linkedreads…

…andscaffolding…



•  Rawdata•  10xChromiummolecule-linkedreads

•  Scaffoldingguide•  Chimerabreakpointdetection

•  Map-anchoredcontigs(fromBauer‘17)•  Preliminaryanchoring,chromosomeassignmentand

chimeradetection•  CSS

•  Chromosomeassignmentandchimeradetection

…andupcoming…


•  Chromosome-ConformationCaptureSequence(Hi-C)•  Fine-scaleorderingandorientationforpseudomolecule

construction

0

300,000,000

600,000,000

900,000,000


Sequ

ence

in b

in (b

p)

EST_zipper_2013

Bauer_2017

NRGene_2018

0

300,000,000

600,000,000

900,000,000


Sequ

ence

in b

in (b

p)

EST_zipper_2013

Bauer_2017

NRGene_2018








…andupcoming…


•  Chromosome-ConformationCaptureSequence(Hi-C)•  Fine-scaleorderingandorientationforpseudomolecule

construction

0e+00

1e+09

2e+09

2 4 6 8log10_scaffold_length

tot_l_in_sizecla

ss assemblyBarke_NRGene

Julius_NRGene

Rye_NRGene

0

300,000,000

600,000,000

900,000,000


Sequ

ence

in b

in (b

p)

EST_zipper_2013

Bauer_2017

NRGene_2018

1G

0.5G

2G

2.5G

1.5G

100 10,000 1,000,000 100,000,000

Wheatcv.Julius

Barleycv.Barke

Rye


NRGeneDeNovoMAGIC3.0Assemblies Rye Wheat(Julius) Barley(Barke)

Totallength(Gbp)(GenomeSize) 6.67(7.9) 14.38(16) 4.18(5.1)

Map-Anchored 6.16 14.20 4.03

N50length(Mbp) 22.49 38.03 38.37

Map-Anchored 24.15 39.36 40.48

Numberofscaffolds 107580 99465 17669

Map-Anchored 1099 3211 522

Proportionsequenceanchored .92 .98 .96

ProportioncompleteBUSCOs .98 .98 .98


Chimericscaffold

Breakpoint!

Achimericscaffold

QualityValidation:Leveragingmolecule-linkedreadsandCSStoidentifychimericscaffolds…

ChromosomeA

ChromosomeB

Identificationby10xmoleculelinkedreads:IdentificationbyCSS:

MappedCSSReads/ContigsChromosom

eofOrig

in

Scaffold


Identificationby10xmoleculelinkedreads:IdentificationbyCSS:

Inreality:Scaffold951

Chromosom

e

Depthofinferred

10xm

olecules

Positioninscaffold(Mbp)


NRGeneDeNovoMAGIC3.0Assemblies Rye Wheat(Julius) Barley(Barke)

Totallength(Gbp)(GenomeSize) 6.67(7.9) 14.38(16) 4.18(5.1)

Map-Anchored 6.16 14.20 4.03

N50length(Mbp) 22.49 38.03 38.37

Map-Anchored 24.15 39.36 40.48

Numberofscaffolds 107580 99465 17669

Map-Anchored 1099 3211 522

Proportionsequenceanchored .92 .98 .96

BadCSSflaggedscaffoldspertenthousand(Number)

4.74(51)

6.24(62)

10.75(19)

Auto-IDdbreaks(10x)perMbp(Number) 0.181(1206) - .0103

(43)


QualityValidation:Assessmentofgenecolinearity…



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scaffold167 scaffold245 scaffold33749 scaffold38 scaffold4680

12

34

56

7

0e+00 3e+07 6e+07 9e+07 0e+00 3e+07 6e+07 9e+07 0e+00 3e+07 6e+07 9e+07 0e+00 3e+07 6e+07 9e+07 0e+00 3e+07 6e+07 9e+07

0e+002e+084e+086e+088e+08

0e+002e+084e+086e+088e+08

0e+002e+084e+086e+088e+08

0e+002e+084e+086e+088e+08

0e+002e+084e+086e+088e+08

0e+002e+084e+086e+088e+08

0e+002e+084e+086e+088e+08

0e+002e+084e+086e+088e+08

pos_scaffold

pos_Hv

Barle

yGe

nomePo

sition

Chromosom

e

RyeScaffoldPosition

RyeScaffold

100millionbp

1billion

bp



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3e+07 6e+07 9e+07pos_scaffold

pos_Hv

Barle

yGe

nomePo

sition

20m

illionbp

RyeScaffold468Position 30millionbp

H.vulgaregenemodels


QualityValidation:Assessmentofgenecolinearity…Confirmationby10xandCSS…

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3e+07 6e+07 9e+07pos_scaffold

pos_Hv

Barle

yGe

nomePo

sition

20m

illionbp

RyeScaffold468Position

H.vulgaregenemodels

RyeScaffoldPosition

30millionbp

30millionbp

Chromosom

eoforig

in

Inferred

coverage

(10X

molecules)

2R1R

3R4R5R6R7R

IlluminaCSSreads








…andupcoming…


•  Chromosome-ConformationCaptureSequence(Hi-C)•  Fine-scaleorderingandorientationfor

pseudomoleculeconstruction


Mascheret.al.2017

ChromosomeConformationCaptureSequencing(Hi-C)

High-densitydistanceinformationformapping/scaffolding

PopSeq

High-densitygeneticmappingonthecheap…

•  Low-coverageWGSdatausedtocallSNPsinassemblyscaffoldsinamappingpopulation

Popu

latio

n

AssemblyScaffolds

GenotypeCallsParentAParentBMissing

Country Institution ScientistGermany IPKGatersleben UweScholzMartinMascher,AndreasHouben,

AndreasBörner,AndreasGraner,NilsSteinJKIGroß-Lüsewitz BerndHackaufJKIQuedlinburg FrankOrdonHMGU KlausMayerKWSLOCHOWGMBH ViktorKorzunHybroSaatzucht JoachimFrommeTUM Bauer

Canada AAC/AAFC AndréLarocheUSASK/GIFS/NRC CurtisPozniac

SharpeKonkinBekkaoui

Poland WestPomeranianUniversityofTechnologySzczecin StefanStojalowskiWarsawUniversityofLifeSciences HannaBolibok-BragoszewskaWarsawUniversityofLifeSciences MonikaRakoczy-Trojanowska

CzechRepublic InstituteofExperimentalBotany JaroslavDoleželFinland UniversityofHelsinki AlanSchulmanUSA TheSamuelRobertsNobleFoundation XuefengMa

KSU JessePolandMSU HikmetBudakUMD VijayKTiwari

UK 2Blades LynneReuberEI Hall

China CAAS JizengJiaSwitzerland ZürichUniversity BeatKellerTurkey CukurovaUniversity HakanÖzkanIsrael NRGene GilRonen

NRGene KobiBaruch


… Thanks!

M.TimothyRabanus-WallaceLeibnizInstituteofPlantGeneticsandCropPlantResearch(IPK)


a whole genome assembly of rye · cv. julius and barley cv. barke) • wgs and mate-pair libraries...

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