everyday de novo diploid assembly

FORRESEARCHUSEONLY.Notforuseindiagnosticprocedures.©10xGenomics,Inc.2016

Everydaydenovodiploid assembly

DeannaM.ChurchOct,2016

@deannachurch

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Disclosures

EmployeeandShareholder

Shareholder

10xGenomics

Personalis

10xGenomicsproductsdescribedareforResearchUseOnly.Notforuseindiagnosticprocedures.

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Acknowledgements

Theentireteamat10x

DavidJaffeNeilWeisenfeldVijayKumarPreyas Shah

PatrickMarks

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Agenda

•Whyhaven’twealwaysdonedenovoassemblyoneverysample?

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Agenda


•WhatareLinked-Reads?

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Agenda


•WhatareLinked-Reads?•Whatdoeseverydaydenovoassemblyenabletoday?

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Whyhaven’twealwaysdonedenovo genomeanalysis?

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Currentapproach:averagingoverhaplotypes

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Averagingoverhaplotypesfailswithincreaseddiversitydoi:10.1038/nature20098

AK1

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Newtechnologyrevealsmoreinformation

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Newtechnologyrevealsmoreinformation

10.1038/nrg3933

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Publichumanassembliestodate

https://www.ncbi.nlm.nih.gov/assembly/organism/9606/latest/

Compositegenomes IndividualGenomes

Hydatidiform moles(singlehaplotypes)

• GRCh38• Celera(2)

• CHM1(9)

• CHM13(5)

• NA12878(9)• HX1• A/JSon• A/JMother

• A/JFather• NA18507• YH1• HS1011• AK1• HuRef

• Lotsoflabor• Lotsoftime• Lotsofcoverage• Lotsofmoney

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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


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MakingLinked-Reads

Longinputmolecule(50Kb)




30xsequence~35fragments~0.2xcoverage

Standardreferencebasedanalysisrecommendations

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MakingLinked-Reads





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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Linked-Readsallowforincreasedphysicalcoverage

150X avg physicalcoverage

Chr13: BRCA2

4/4/2016 Loupe

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

▲

쁛 ►

>56X avgread coverage(assembly)

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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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Assemblymadeeasy

FASTABCL SupernovaDenovoAssembly

1200MNA19240

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

1server348Gbmemory2dayscompute

1library1nginput

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Assemblymadeeasy


1200MNA192401library

1nginput


1server(28cores)348Gbmemory2dayscompute

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Assemblymadeeasy


1200MNA192401library

1nginput


1server(28cores)348Gbmemory2dayscompute

megabubble megabubble megabubble

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Performanceovermultiplehumansamples


sample ethnicity sex cov frag

N50contig(Kb)

N50scaffold(Mb)

N50Phaseblock(Mb)

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)


00.51

1.52

2.53

3.54

4.55

500 700 900 1,100 1,300

PhaseBlockN50

(Mb)


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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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Comparisontotruthdata

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Assemblyassessment

Supernova10x Othermethods

0

5

10

15

20

25

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

PercentGRCh37100mersmissingperassembly

Missing100mershaploid Missing100mersdiploid

Diploid Haploid

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Whatdoeseverydaydenovoassemblyenable?

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Ideal:Completegenomeinformation

doi:10.1038/nature09534

• SNVs• Deletions• Insertions• Inversions• Translocations

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Areasinwhichassemblyexcels:diverseregions

AluY

Supernova(denovo)

PacBio Reads

IlluminaReads

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Areasinwhichassemblyexcels:insertions

Supernova(denovo)

PacBioReads

IlluminaReads

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SHANK2

GRCh37:chr11

GRCh37.p13:chr11_fix_patch

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SHANK2

GRCh37:chr11

GRCh37.p13:chr11_fix_patch35kb

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Hap1_scaffold7938

Hap2_scaffold7939

chr11

SHANK2

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Hap1_scaffold7938

Hap2_scaffold7939

chr11

SHANK2

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Hap1_scaffold7938

Hap1_scaffold7939

chr11

SHANK2

chr11

Hap2_scaffold7939

SHANK2

Hap1_scaffold7938

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Assemblyanalysis:alignmentworkneeded

SHANK2

Supernova(denovo)

PacBio Reads

IlluminaReads

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Areasinwhichassemblyexcels:inversions

GRCh37 chrX:6137041-6138541 (NLGN4X)

Supernova(denovo)

PacBio Reads

IlluminaReads

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Assemblyanalysis:alignmentworkneeded

GRCh37 chrX:6137041-6138541 (NLGN4X)

Hap1_scaffold5127

Hap2_scaffold5128

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Fasta isalossy format

megabubble megabubble megabubble

multi-Mbphaseblocks

manyMbscaffold

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

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Nativeformatshavemoreinformation

Supernova(denovo)

LongRangerReferencebased

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Supernova(denovo)


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Supernova(denovo)


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Conclusions

•Routine,denovo,diploidassemblyof1000sofsamplesispossibletoday!

•Earlyuseswillbeforbetterresolutionofdivergentregionsandnovelsequence

•Anewgenerationoftoolsneedstobedevelopedtofullyutilizeassemblydata

everyday de novo diploid assembly

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