FORRESEARCHUSEONLY.Notforuseindiagnosticprocedures.

Everydaydenovoassembly

GRCAssemblyWorkshopatGenomeInformatics

DeannaM.ChurchSeniorDirectorofApplicationsSep19,2016

@deannachurch

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Acknowledgements

Theentireteamat10x

DavidJaffe

NeilWeisenfeld

VijayKumar

Preyas Shah

NCBI:

FrancoiseThibaud-Nissen

ValerieSchneider

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Disclosures

EmployeeandShareholder

Shareholder

10xGenomics

Personalis

10xGenomicsproductsdescribedareforResearchUseOnly.Notforuseindiagnosticprocedures.

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Questionsfromtheorganizers

Arenewassembliesusingthereference?Cantheyhelpmakethereferencebetter?Dotheymakethereferenceobsolete?

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Agenda

Whyhaven’twealwaysdonedenovogenomeanalysis?

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Agenda

Whyhaven’twealwaysdonedenovogenomeanalysis?WhatareLinked-Reads?

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Agenda

Whyhaven’twealwaysdonedenovogenomeanalysis?WhatareLinked-Reads?HowdoLinked-Readsenableeverydaydenovoassembly?

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Whyhaven’twealwaysdonedenovo genomeanalysis?

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KellyHowe,LawrenceBerkeleyLaboratory

KellyHowe,LawrenceBerkeleyLaboratory

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ReferencequalityisHARD

DOI:10.1038/nature03001

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Ouractualgenome:diploid

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Howwerepresentourgenome:haploid

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Currentapproach:averagingoverhaplotypes

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Currentapproach:averagingoverhaplotypes

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Currentapproach:averagingoverhaplotypes

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Currentapproach:averagingoverhaplotypes

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Problem:bothallelesdifferfromeachother

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WhatareLinked-Reads?

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Unlinked-Reads:shortrangeinformation

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Linked-Reads:longrangeinformation

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StartwithlongmoleculesNA19240

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MakingLinked-Reads

P5 16bpBCR1 Nmer gDNA Insert

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MakingLinked-Reads

Longinputmolecule

Excessofsequenceableinsertsrandomlyprimedoffeachlongmolecule

P5 16bpBCR1 Nmer gDNA Insert

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MakingLinked-Reads

Longinputmolecule(50Kb)

Excessofsequenceableinsertsrandomlyprimedoffeachlongmolecule

P5 16bpBCR1 Nmer gDNA Insert

Longinputmolecule(50Kb)

30xsequence~35fragments~0.2xcoverage

Standardreferencebasedanalysisrecommendations

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MakingLinked-Reads

Longinputmolecule(50Kb)

Excessofsequenceableinsertsrandomlyprimedoffeachlongmolecule

P5 16bpBCR1 Nmer gDNA Insert

Longinputmolecule(50Kb)

56xsequence~65fragments~0.4xcoverage

Supernovaanalysisrecommendations

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SyntheticLongReads:lessphysicalcoverage

CA B

SequencingcostPhysicalcoverage

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Linked-Reads:greaterphysicalcoverage

CA B

SequencingcostPhysicalcoverage

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Example– MoleculevsReadCoverage

150X avgmolecule coverage

Agivengenomiclocuswillhave

150X avg moleculedepth,and30X avg readdepth

(150Xmoleculedepth)x (0.2Xread/m)=30Xreaddepth

Chr13: BRCA2

4/4/2016 Loupe

http://loupe.fuzzplex.com/loupe/view/MTk1MzgtUEhBU0VSX1NWQ0FMTEVSX1BELTEwMTMuMC4yNi5sb3VwZQ==/reads?ranges=chr13%2B32850000-chr1… 1/1

▲

쁛 ►

>30X avgread coverage

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GeneratingLinked-Reads

Startwith:

HMWgDNA,100Kb+molecules1.0ng inputDNA=300copiesofthegenome

0.5ngDNA=150 copiesofthegenome,partitionedinto>1MGEMs

DNA

OilBarcodedPrimerLibrary Enzyme Collect

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HowdoLinked-Readsenableeverydaydenovoassembly?

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Assemblymadeeasy

FASTABCL SupernovaDenovoAssembly

1200MNA19240

http://www.biorxiv.org/content/early/2016/08/19/070425

1server348Gbmemory2dayscompute

1library1.25nginput

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Assemblymadeeasy

FASTABCL SupernovaDenovoAssembly

1200MNA192401library

1.25nginput

http://www.biorxiv.org/content/early/2016/08/19/070425

1server(28cores)348Gbmemory2dayscompute

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Assemblymadeeasy

Measure ValueNumberof scaffolds>=10Kb 1.17 KEdgeN50 17.45KbContig N50 118.8KbPhaseblock N50 9.3MbScaffoldN50 16.4Mb

FASTABCL SupernovaDenovoAssembly

1200MNA19240

http://www.biorxiv.org/content/early/2016/08/19/070425

1server(28cores)348Gbmemory2dayscompute

1library1.25nginput

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Performanceovermultiplesamples

http://www.biorxiv.org/content/early/2016/08/19/070425

sample ethnicity sex cov frag N50contig

N50scaffold

N50Phaseblock

gap

NA19238 YRI F 56 115 114.6 18.7 8 2.1

NA19240 YRI F 56 125 118.8 16.4 9.3 2.3

HG00733 PR F 56 106 123.6 17.8 3.4 2.0

HG00512 HAN M 56 102 113.2 15.4 2.7 2.2

NA24385 AJ M 56 120 106.4 15.1 4.2 2.6

HGP EUR M 56 139 120.2 18.6 4.5 2.5

NA12878 EUR F 56 92 118.5 16.4 2.8 2.9

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HighqualityAssemblyatlowercoverage

102104106108110112114116118120122

500 700 900 1,100 1,300

ContigN50

(kb)

Numberofreads(millions)

0

5

10

15

20

25

500 700 900 1,100 1,300

ScaffoldN50

(Mb)

Numberofreads(millions)

0

1

2

3

4

5

500 700 900 1,100 1,300PhaseBlockN50

(Mb)

Numberofreads(millions)

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DeNovoPerformanceDrasticallyImproveswithIncreasedDNALength

020,00040,00060,00080,000100,000120,000

0 10,000 20,000 30,000 40,000 50,000 60,000

ContigN50

0

5

10

15

20

0 10,000 20,000 30,000 40,000 50,000 60,000

ScaffoldN50

(Mb)

0100,000200,000300,000400,000500,000

0 10,000 20,000 30,000 40,000 50,000 60,000PhaseBlock

N50

DNALength

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SupernovaAssembler

stuff

separateassembliesofhomologousloci

http://www.biorxiv.org/content/early/2016/08/19/070425

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Assemblyarchitecture=phaseblocks

megabubble megabubble megabubble

multi-Mbphaseblocks

manyMbscaffold

microstructure• bubbles,oftenatindeterminatepoly-A• shortgaps,oftenatpoly-A

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Assemblyassessment

Supernova10x Othermethods

0

5

10

15

20

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NA19238 NA19240 HG00733 HG00512 NA24385 HGP NA12878 YH NA12878 NA12878 NA12878 NA24385 NA24143

PercentGRCh37100mersmissingperassembly

Missing100mershaploid Missing100mersdiploid

Diploid Haploid

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Comparisontotruthdata

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Improvingthereferenceassembly?

GRCh38:chr6(NC_000006.12

NA12878,hap0,scaf.21653(prev1.1)

260Kbofnewsequence

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Bettergenotypereconstruction

chrX:6,219,000-6,220,500(GRCh38)NLGN4X(neuroligin 4,x-linked)

43chrX:6,218,359-6,221,000(GRCh38)

Bettergenotypereconstruction

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Questionsfromtheorganizers

Arenewassembliesusingthereference?

Supernova:denovoassemblyDiploidreconstruction

NOYes

Assemblyconstruction

Assemblyanalysis

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Questionsfromtheorganizers

Cantheyhelpmakethereferencebetter?

Yes

Supernova:individualgenomereconstructionContributingnewsequencestopopulationgraph

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Questionsfromtheorganizers

Dotheymakethereferenceobsolete?

NO

Supernova:NotreferenceassembliesBetterindividualgenomereconstruction

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Thanks!