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AWholeGenomeAssemblyofRye(Secalecereale)M.TimothyRabanus-WallaceLeibnizInstituteofPlantGeneticsandCropPlantResearch(IPK)
MTRabanus-Wallace2018
MTRabanus-Wallace2018
Whyrye?
MTRabanus-Wallace2018
QTLanalysisforSurvivalAfterWinter(SAW)scoresinrye:
Erathetal.2017
Frost-damageinwinterwheatPhoto:IngridKristjanson(http://cropchatter.com/impact-of-frost-on-winter-wheat-fall-rye/)
MTRabanus-Wallace2018
RyeSecalecerealeChallenge1)Length(7.9Gbp)
Assemblychallenges…
MTRabanus-Wallace2018
RyeSecalecerealeChallenge1)Length(7.9Gbp)Challenge2)90+%repetitive
Assemblychallenges…
MTRabanus-Wallace2018
RyeSecalecerealeChallenge1)Length(7.9Gbp)Challenge2)90+%repetitiveChallenge3)Obligateoutcrossing
Assemblychallenges…
MTRabanus-Wallace2018
AssemblyScaffolds
MTRabanus-Wallace2018
AssemblyScaffolds
MolecularMap
MTRabanus-Wallace2018
AssemblyScaffolds
MolecularMap
Genome
MTRabanus-Wallace2018
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
Martis‘13:ARyeProto-Genome(“Zipper”)• RNAseq(ExpressedSequenceTags;ESTs)
• Rawsequenceinformation• ChromosomeSurveySequencing(CSS)
• Chromosome-assignedsequenceinformation• 5KSNP-array-basedgeneticmap
• ESTanchoringbackbone• Interspeciesgenecolinearity
• Fine-scaleorderingandgeneidentificationbysequencehomology
MTRabanus-Wallace2018
Martis‘13:ARyeProto-Genome(“Zipper”)• RNAseq(ExpressedSequenceTags;ESTs)
• Rawsequenceinformation• ChromosomeSurveySequencing(CSS)
• Chromosome-assignedsequenceinformation• 5KSNP-array-basedgeneticmap
• ESTanchoringbackbone• Interspeciesgenecolinearity
• Fine-scaleorderingandgeneidentificationbysequencehomology
MTRabanus-Wallace2018
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
Martis‘13:ARyeProto-Genome(“Zipper”)• RNAseq(ExpressedSequenceTags;ESTs)
• Rawsequenceinformation• ChromosomeSurveySequencing(CSS)
• Chromosomeassignment• 5KSNP-array-basedgeneticmap
• ESTanchoringbackbone• Interspeciesgenecolinearity
• 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
Martis‘13:ARyeProto-Genome(“Zipper”)• RNAseq(ExpressedSequenceTags;ESTs)
• Rawsequenceinformation• ChromosomeSurveySequencing(CSS)
• Chromosomeassignment• 5KSNP-array-basedgeneticmap
• ESTanchoringbackbone• Interspeciesgenecolinearity
• Fine-scaleESTorderingBauer‘17:ADraftGenome• WGSandMate-PairLibraries
• Rawsequenceandhierarchicalscaffolding• CSS
• Contigandmate-pairassignmentpre-scaffolding
• High-densitySNPmap(iSelectRye600kArray)• Toanchorscaffolds
• DArTseqgeneticmap• Toguidescaffoldinganddetectchimeras
• Martis’13genomezipper(updated)MTRabanus-Wallace2018
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
Martis‘13:ARyeProto-Genome(“Zipper”)• RNAseq(ExpressedSequenceTags;ESTs)
• Rawsequenceinformation• ChromosomeSurveySequencing(CSS)
• Chromosomeassignment• 5KSNP-array-basedgeneticmap
• ESTanchoringbackbone• Interspeciesgenecolinearity
• Fine-scaleESTorderingBauer‘17:ADraftGenome• WGSandMate-PairLibraries
• Rawsequenceandhierarchicalscaffolding• CSS
• Contigandmate-pairassignmentpre-scaffolding
• High-densitySNPmap(iSelectRye600kArray)• Toanchorscaffolds
• DArTseqgeneticmap• Toguidescaffoldinganddetectchimeras
• Martis’13genomezipper(updated)MTRabanus-Wallace2018
0
300,000,000
600,000,000
900,000,000
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
Martis‘13:ARyeProto-Genome(“Zipper”)• RNAseq(ExpressedSequenceTags;ESTs)
• Rawsequenceinformation• ChromosomeSurveySequencing(CSS)
• Chromosomeassignment• 5KSNP-array-basedgeneticmap
• ESTanchoringbackbone• Interspeciesgenecolinearity
• Fine-scaleESTorderingBauer‘17:ADraftGenome• WGSandMate-PairLibraries
• Rawsequenceandhierarchicalscaffolding• CSS
• Contigandmate-pairassignmentpre-scaffolding
• High-densitySNPmap(iSelectRye600kArray)• Toanchorscaffolds
• DArTseqgeneticmap• Toguidescaffoldinganddetectchimeras
• Martis’13genomezipper(updated)MTRabanus-Wallace2018
0
300,000,000
600,000,000
900,000,000
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
Bauer_2017
MTRabanus-Wallace2018
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…
MTRabanus-Wallace2018
2018:ApproachingReferenceQualityAnNRGeneDeNovoMAGIC3.0assembly(analoguesinwheatcv.Juliusandbarleycv.Barke)• WGSandmate-pairlibraries
• Rawdata• 10xChromiummolecule-linkedreads
• Scaffoldingguide• Chimerabreakpointdetection
• Map-anchoredcontigs(fromBauer‘17)• Preliminaryanchoring,chromosomeassignmentand
chimeradetection• CSS
• Chromosomeassignmentandchimeradetection
…andupcoming…
• PopSeqhigh-densitygeneticmapping• Mapanchoringandchimeradetection
• Chromosome-ConformationCaptureSequence(Hi-C)• Fine-scaleorderingandorientationforpseudomolecule
construction
0
300,000,000
600,000,000
900,000,000
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
Bauer_2017
NRGene_2018
0
300,000,000
600,000,000
900,000,000
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
Bauer_2017
NRGene_2018
MTRabanus-Wallace2018
2018:ApproachingReferenceQualityAnNRGeneDeNovoMAGIC3.0assembly(analoguesinwheatcv.Juliusandbarleycv.Barke)• WGSandmate-pairlibraries
• Rawdata• 10xChromiummolecule-linkedreads
• Scaffoldingguide• Chimerabreakpointdetection
• Map-anchoredcontigs(fromBauer‘17)• Preliminaryanchoring,chromosomeassignmentand
chimeradetection• CSS
• Chromosomeassignmentandchimeradetection
…andupcoming…
• PopSeqhigh-densitygeneticmapping• Mapanchoringandchimeradetection
• 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
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
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
MTRabanus-Wallace2018
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
MTRabanus-Wallace2018
Chimericscaffold
Breakpoint!
Achimericscaffold
QualityValidation:Leveragingmolecule-linkedreadsandCSStoidentifychimericscaffolds…
ChromosomeA
ChromosomeB
Identificationby10xmoleculelinkedreads:IdentificationbyCSS:
MappedCSSReads/ContigsChromosom
eofOrig
in
Scaffold
MTRabanus-Wallace2018
Identificationby10xmoleculelinkedreads:IdentificationbyCSS:
Inreality:Scaffold951
Chromosom
e
Depthofinferred
10xm
olecules
Positioninscaffold(Mbp)
MTRabanus-Wallace2018
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)
MTRabanus-Wallace2018
QualityValidation:Assessmentofgenecolinearity…
MTRabanus-Wallace2018
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
MTRabanus-Wallace2018
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
MTRabanus-Wallace2018
QualityValidation:Assessmentofgenecolinearity…
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5.2e+08
5.4e+08
5.6e+08
5.8e+08
3e+07 6e+07 9e+07pos_scaffold
pos_Hv
Barle
yGe
nomePo
sition
20m
illionbp
RyeScaffold468Position 30millionbp
H.vulgaregenemodels
MTRabanus-Wallace2018
QualityValidation:Assessmentofgenecolinearity…Confirmationby10xandCSS…
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5.2e+08
5.4e+08
5.6e+08
5.8e+08
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
MTRabanus-Wallace2018
2018:ApproachingReferenceQualityAnNRGeneDeNovoMAGIC3.0assembly(analoguesinwheatcv.Juliusandbarleycv.Barke)• WGSandmate-pairlibraries
• Rawdata• 10xChromiummolecule-linkedreads
• Scaffoldingguide• Chimerabreakpointdetection
• Map-anchoredcontigs(fromBauer‘17)• Preliminaryanchoring,chromosomeassignmentand
chimeradetection• CSS
• Chromosomeassignmentandchimeradetection
…andupcoming…
• PopSeqhigh-densitygeneticmapping• Mapanchoringandchimeradetection
• Chromosome-ConformationCaptureSequence(Hi-C)• Fine-scaleorderingandorientationfor
pseudomoleculeconstruction
MTRabanus-Wallace2018
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
MTRabanus-Wallace2018
… Thanks!
M.TimothyRabanus-WallaceLeibnizInstituteofPlantGeneticsandCropPlantResearch(IPK)
MTRabanus-Wallace2018