Medical genetics:

Identification of hidden structural variants

with long-read sequencing

Alexander HoischenAssistant Professor Immuno-Genomics

Scientific Director Radboud Genomics Technology Center

Departments of Human Genetics and Internal MedicineRadboud University Medical Center,

Nijmegen, The Netherlands

Contact: alexander.hoischen@radboudumc.nlwww.radboudumc.nl/en/immunogenomics

@ahoischen

Engineer/PhD-student/Postdocjobs in bioinformatics available!

Full Disclosure

This project is a collaborationbetween RUMC and PacBio Inc. in which reagents costs were shared.

Finding the answer in the genome

6 billion nucleotides46 chromosomes

2 people differ at >4 million positions

1 variant (mutation) can result in disease*

Genome sequencing: All variants in one experiment!

* With all variant types known to cause disease: karyotype aberrations, SVs, indels, SNVs

De novo mutations in ID• Intellectual disability (ID = IQ <70) is a model for severe, sporadic disorders

• Similar to autism spectrum disorder (ASD), epilepsy and other (neuro-) developmental disorders (NDDs)

• >60% of severe ID is caused by de novo mutations

• De novo mutation rate for SNVs is ca. 1.8x10-8, i.e. 30-100 de novo SNVs per

genome per generation (i.e. 1-2 per exome); • De novo mutation rate for large CNVs ca. 0.2 per generation• De novo mutation rate for SVs/large indels – largely unknown!

Nat Rev Genet. 2012 Jul 18;13(8):565-75

De novo mutations reduce genome complexity/wealth of variation greatlySevere, sporadic diseases offer opportunity to identify novel paradigms

Gilissen et al. Nature 2014

De Ligt et al. NEJM 2012

De Vries et al. AJHG 2006& Vulto-van Silfhoutet al. Hum Mut 2013

New Genomic Technologies elucidate Intellectual Disability

Intellectual disability

42%3

11.6%1

27%2

Genomic microarray

Exome sequencing

2014Whole genome

sequencing

Single gene test~1-5%

% of ID patients with a

diagnosis

62%

±1,500 ID patients

Nodiagnosis

De novoSNVs

De novoSVsInherited

1Vulto-van Silfhout et al. Hum Mutat. 2013; 2De Ligt et al. NEJM. 20123Gilissen et al. Nature 2014

Majority is de novo!

2012

2008 38%

...hidden SVs – i.e. long reads?

Hidden de novo SVs in unsolved ID trios?

Patient cohort:

• A clinically well-characterized patient population with intellectual disability. These samples have been previously analyzed extensively:• CNV-microarrays (Vulto-van Silfhout et al. Hum Mut 2013)

• Whole exome sequencing (de Ligt et al. NEJM 2012)

• Short-read whole genome sequencing (Gilissen et al. Nature 2014)

• NovaSeq 30-40x whole genome sequencing

• All previous analyses failed to detect a causal variant

This study:

• Here we perform long-read SMRT sequencing on the Sequel platform in 5 such patient-parent trios

Hypothesis:

• Hidden, previously undetected, de novo SVs may explain disease

DNA• 5 trios

• Fresh gDNA from whole blood• Final libraries: fragment sizes of 40-70kb

Genomic DNAs Sheared DNAs

Michael Kwint

Our first data

5 first trios: • 1 trio with ~40x sequenced in Menlo Park (PacBio)• 4 trios with ~15x in Nijmegen

• Output:• On average 4.7Gb/SMRT cell*• 11.6kb average read length*

• All trios were also sequenced by short-read WGS (Complete Genomics, 80x & Illumina NovaSeq, 40x)

• BioNano mapping data has been generated for one trio

*Sequel 2.0 chemistry with mixture of 4.0 and 5.0 software.

New developments, latest WGS samples

Express library:

• Lower input, higher yield, less hands-on; amenable to automation

• 25 SMRT cells:• Average read length 19kb• Average output Gb per SMRT cell:

6.8Gb (Max. 9.2Gb)

Michael Kwint

Gigabase output – 234 SMRT cells

Sequel 2.0 chemistry with mixture of 4.0 and 5.0 software.

10X

20X

0

20

40

60

80

100

120

s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 s11 s12 s13 s14 s15Samples

Base

s (

Gb) Trio

Trio1

Trio3

Trio4

Trio5

Trio7

Mapped bases per sample

Coverage comparison with NovaSeq short reads

• 28Mb of the human reference genome is only covered reliably

by long reads;

• 12Mb overlaps with genic regions;

• 757kb coding sequence

0

5

10

15

20

25

30

Trio5 S1 Trio5 S2 Trio5 S3

Me

ga b

ase

s

Genomic

Genic

Exonic

SV variant calling

• Solo vs. joint calling using pbsv caller (early access by PacBio) • Joint calling: call the three samples of one trio together

Number of SVs/genome

0

5,000

10,000

15,000

20,000

25,000

30,000

Trio1 S1 Trio1 S2 Trio1 S3

Solo calling

Joint calling

Initial results at ±6x coverage:

[n]

Number of SVs (>50bp)

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

Trio1 Trio3 Trio4 Trio5 Trio7

Solo

Joint

Trio 1 Trio 2 Trio 3 Trio 4 Trio 5

[n]

Structural variants: 50bp – 1kb

0

5000

10000

15000

20000

25000

Insertions

Deletions

Alu insertions and deletions

[n]

[size bp]

SVs: 1kb – 10kb

0

200

400

600

800

1000

1200

1400

1600

INS

DEL

LINE elements

[n]

[size bp]

SV comparison with 40x NovaSeq

• ~70% novel SVs:Of >25,000 SVs – ca. 17,500 are novel; only ~30% of SVs also called in NovaSeq data using Manta*

• ~80% of insertions are novel, ~55% of deletions are novel (i.e. not called in NovaSeq data)

• There are ~1,000 SVs that are called by NovaSeq data only (~100 insertions; ~900 deletions)

*Overlaps done using Survivor: https://github.com/fritzsedlazeck/SURVIVOR

Variants missed by short read sequencing

PacBio 40x child

PacBio 40x father

PacBio 40x mother

NovaSeq 40x child

116bp heterozygous deletion

Paternally inherited

Not called.Ambigiuous reads?

Variants missed by short read sequencing

PacBio 40x child

PacBio 40x father

PacBio 40x mother

NovaSeq40x child

334bp insertion

Maternallyinherited

Not called.

• Comparison with 40X WGS data (NovaSeq, called with Manta**): Using SURVIVOR*

How many SVs are novel?

*https://github.com/fritzsedlazeck/SURVIVOR**https://github.com/Illumina/manta/

15,430

(68.8%)

5,897

(26.3%)

1,092

(4.8%)60% in simple

repeats >100 bp81% in simplerepeats >100 bp

35% in simple

repeats >100 bp

Structural variants ≥50 bp PacBio vs Illumina NovaSeq

SV distribution over samples

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Co

un

t o

f SV

s

Individuals with respective SV

SVs >50bp

common/reference issues

rare/private

Filter for de novo SVs – value of trios

Manual curation/high qual.: ~5-10 candidate de novo SV per patient

~40 SVs not in

parents

~1000 SVs not in

population

All ~25,000

SVs

De novo calling of SVs: DNM shortlist

Trio1 Trio3 Trio4 Trio5 Trio7

DEL INS INV DEL INS INV DEL INS INV DEL INS INV DEL INS INV0

5

10

15

# d

en

ovo

ca

nd

ida

tes

Lenient

Strict

De novo candidatesTrio 1 Trio 2 Trio 3 Trio 4 Trio 5

Chr16: 65bp deletion intragenic KLHDC4

PacBio 17x child

PacBio 20x father

PacBio 17x mother

De novo candidate SVs• 554bp deletion in intergenic region

PacBio 15x child

PacBio 15x father

PacBio 15x mother

544bp deletion

Validations for de novo candidates are ongoing

Last example: 544bp intergenic deletion

Conclusion:This is a true 544bp deletion

But it is inherited from the healthy father

wt allele (1210bp)

deletion allele 666bp)

patient father mother

Validations so far

• >30 validations; only one FP; all inherited so far

• No validated de novo SV in any patient yet

• In some regions PCR-primer design challenging

Other SVs: Indels? Inversions?

• How many smaller events can we find?• Indels 20-50bp

• How many inversions per genome?

trio

1_f

ath

er

trio

1_m

oth

er

trio

1_p

atie

nt

trio

2_f

ath

er

trio

2_m

oth

er

trio

2_p

atie

nt

trio

3_m

oth

er

trio

3_p

atie

nt

trio

3_f

ath

er

trio

4_p

atie

nt

trio

4_f

ath

er

trio

4_m

oth

er

trio

5_f

ath

er

trio

5_m

oth

er

trio

5_p

atie

nt

SVs/indels >20bp

>33-40,000 SVs/genome (20-50bp)

PBSV caller with lower cut-off of 20bp

0

10,000

20,000

30,000

40,000

50,000

60,000

70,000

80,000

Solo

Joint

How many indels are novel?

*https://github.com/fritzsedlazeck/SURVIVOR**https://github.com/Illumina/manta/

21,261

(57.9%)

11,961

(32.6%)

3,506

(9.5%)34% in simple

repeats >100 bp73% in simplerepeats >100 bp

26% in simple

repeats >100 bp

Indels 20-50bp PacBio vs Illumina NovaSeq

• Comparison with 40X WGS data (NovaSeq, called with Manta**): Using SURVIVOR*

SV distribution over samples

rare/private

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Co

un

t o

f SV

s

Individuals with respective SV

SVs > 20bp

How many are inherited?

Up to 74% in patient& parent (18% random)

Inversions in 15 genomes

0

50

100

150

200

250

Child Father Mother Child Father Mother Child Father Mother Child Father Mother Child Father Mother

Trio1 Trio2 Trio3 Trio4 Trio5

Called with PBSV developers version

Inversions – size distribution

0

50

100

150

200

250

300

0-100 100-200 200-300 300-400 400-500 >500

Inversions <1Kb, bin=100 bp

0

2

4

6

8

10

12

14

Inversions 1-10 Kb, bin=1 Kb

Next steps

• Are any candidate de novo SVs truly de novo?

• If they are, could they explain disease?• Genotype/phenotype recurrence in other cases?

• Do rare hidden SVs unmask recessive disease?

Extra ‘goodies’ of long-reads?

• Can we detect de novo SNVs?

• Phasing de novo mutations

• Phasing of candidate comp. het. variants

We can detect de novo SNVs

PacBio 40x child

PacBio 40x father

PacBio 40x mother

de novo c. 1685A>C; p.(His562Pro); TBKBP1

We can phase de novo SNVsde novo c. 1685A>C;

p.(His562Pro); TBKBP1

PacBio 40x child

PacBio 40x father

PacBio 40x mother

On same allele: Maternalheterozygous SNP

Phasing de novo mutations important to understand DNM biology

e.g.: Goldmann et al. Nat Genet 2016 & 2018

Compound heterozygous variants?

Allele 1Heterozygous variant

PacBio 10x childonly

Allele 2Heterozygous variant

Work in progress..

• Calling SV with other tools, e.g. sniffles• Calling single nucleotide variants• Assembly using the GRCh38 reference and full de novo assembly• Comparison with other technologies (10x genomics, bionano, etc.)

Summary

• Per genome: High quality coverage for 28Mb of previously uncovered sequence

• SMRT sequencing allows detection of ~25,000 SVs per genome

• Also: >33,000 indels (20-50bp) are called per genome

• Majority of those SVs/indels were not detected by short-read WGS

• Long reads to comprehend de novo mutation rates of indels/SVs – start to understand clinical relevance

PacBio in diagnostics

• Kornelia Neveling/Marcel Nelen:

Use long-range PCR for complex human regions:• HLA (5 amplicons), collab. with medical immunology• Pseudogenes• mtDNA• Repeat expansions

Launch of diagnostic PacBio assay for HLA: June 15th! ...others will follow in 2018

Full list of diagnostic tests: www.genomediagnosticsnijmegen.nl

EU: 15Mio€ H2020 grant – start in 2018

Ambition to solve a significant number of rare diseases (RD) that remained unsolved after exome/genome sequencing

Total RD cases ~20,000

Aim for >500 long-read genomes & >100 full-isoform seqs

Solving the unsolved Rare Diseases

www.SOLVE-RD.eu*Co-coordinated by: Tübingen, Leicester, Nijmegen

Goal:Latest genomics tools &

integrating other omics to understand biology and disease

https://www.x-omics.nl

https://www.solve-rd.eu

Acknowledgements

Radboud UMC

• Michael Kwint• Marc Pauper• Maartje van der Vorst

• Jordi Corominas Galbany• Marcel Nelen• Kornelia Neveling

• Christian Gilissen• Lisenka Vissers• Han Brunner

All patients and families

PacBio• Aaron M Wenger

• Primo Baybayan • Luke Hickey• Jonas Korlach

• Kevin Corcoran

PacBio EU

John Kuijpers, Gerard van de Burgt, Ralph

Vogelsang, David Stucki, Philip Lobb, John

Baeten, Gerrit Kuhn, Deepak Singh