The impact of next generation sequencing on human genetics

Prof. dr. Frans P.M. Cremers

Department of Human Genetics, Nijmegen, the Netherlands

S1 student presentation, Cebior, Semarang, 25 July 2010

Milestones in molecular genetics

• 1953: Watson, J. & Crick, F.: The double helix

• 1977: Sanger, F. et al.: DNA sequencing

• 1983: Mullins, K.B.: Polymerase Chain Reaction

• 2005: Margulies, M. et al. and many others: Next generation sequencing

Sanger sequencing: technique

3

ATGCTTCGGCAAGA

ATGC

ATGCT

ATGCTT

ATGCTTC

1

2

5

4

3

Gene X

exon

exon

exon

exon

exon

PCR amplification

3 TACGAAGCCGTTCT

ATG

DNAtemplate

33

3

33

CC

T

T

T

T

C CA GG

GA

A

CT

TACGAAGCCGTTCT

ATG

C

TT

C

Selection on size

ABI3730

Primers

Sanger sequencing: costs

• 48 electrophoresis capillaries• 500 nucleotides per capillary

~25.000 nucleotides per run

Costs: € 5 per capillary = € 250 / 25.000 nt

€ 0.01/nt = Rp. 100/nt

Sanger sequencing: applications

Human Genetics:• DNA Diagnostics: sequencing known disease genes (e.g. cystic fibrosis, retinoblastoma)

• Searching for new genes: analysis of candidate genes in genetic linkage interval

Genes: on average 10 exons that encode for the protein

ATG TAATGATAG

Translationstop

(translation startcodon for

Methionine)

Protein

6

7

Disease # GenesSanger

sequencing costs

Hereditary breast cancer 2€ 500

Rp. 5.000.000

Ataxia ~10€ 2.500

Rp. 25.000.000

Hereditary blindness ~100€ 25.000

Rp. 250.000.000

Mental retardation ~1000€ 250.000

Rp. 2.500.000.000

Sanger sequencing: limitations when testing diseases with large genetic heterogeneity

DNA-Enrichment by array sequence capture:

1. DNA fragmentation

2. Hybridization to synthesized probes

Next generation sequencing (NGS)

5. Sequencing

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3. Stringent washing

4. Elution & amplification

Library preparation Emulsion-PCR

Pyrosequencing

NGS: Massive parallel sequencing (Roche 454)

NGS: 1000-fold increase in output

• 1 million parallel reads• 500 bp per read

500,000,000 nt

• 20 x coverage needed

• Effective: 25.000.000 nt

Sanger sequencing (ABI 3730): 25.000 nt

NGS: 100-fold cheaper

NGS: € 2.500 / 25.000.000 nt

€ 0.0001 / nt (Rp. 1 / nt)

Sanger sequencing: € 0.01 / nt (Rp. 100 / nt)

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Disease # GenesSanger

sequencing costs

NGScosts

Hereditary breast cancer

2€ 500

Rp. 5.000.000€ 5

Rp. 50.000

Ataxia ~10€ 2.500

Rp. 25.000.000€ 2.5

Rp. 250.000

Hereditary blindness

~100€ 25.000

Rp. 250.000.000€ 250

Rp. 2.500.000

Mental retardation

~1000€ 250.000

Rp. 2.500.000.000€ 2.500

Rp. 25.000.000

Molecular Diagnostics:Sanger sequencing vs NGS

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Disease # GenesNGS

costs

Hereditary breast cancer 2€ 0.5

Rp. 50.000

Ataxia ~10€ 2.5

Rp. 250.000

Hereditary blindness ~100€ 250

Rp. 2.500.000

Mental retardation ~1000€ 2.500

Rp. 25.000.000

NGS, application 1: identifying defects in known disease genes

NGS, application 2: identifying genetic defect in genomic region

Identification of a new gene for familial exudative vitreoretinopathy

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Nikopoulos K. et al. Am J Hum Genet. 86:240-247, 2010.

Familial exudative vitreoretinopathy

Fundus:• avascular zone

Familial exudative vitreoretinopathy

Fundus:• avascular zone• retinal detachment• “stretched/dragged”

vasculature

Visual acuity:• normal blindness

Linkage at chromosome 7

7 7

LOD LOD

0.50

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0.000.501.001.502.002.503.003.50

chr chr 7

4.00

Candidate gene analysis

340 genes

126.4 Mb109.7 Mb

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Position reference

alleleRef. allele

Variant allele

Total# of

reads

# of variant reads

% variantreads

Ref. amino acid

Variant amino acid Gene

PhyloP

score120216091 C G 20 10 50 A P TSPAN12 5.3298870495 G A 26 16 62 R C PTCD1 3.06

100209410 G A 15 8 53 R H ZAN 1.8199835402 C T 13 6 46 P L PILRA 1.75

113306419 C T 15 6 40 S N PPP1R3A 1.05100473466 A G 38 13 34 T A MUC17 0.60128099699 C G 7 5 71 I M FAM71F2 0.42115411632 C T 14 5 36 D N TFEC -0.45

Candidate gene analysis

PhyloP score: conservation of a nucleotide on a given sequence among 44 vertebrate species.

NGS of 330 genes in 40 Mb region

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Identification of a new gene for familial exudative vitreoretinopathy

21

TSPAN12 p.Ala237Pro

c.709G>C

NGS, application 3: sequencing of whole genomes

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• Analysis of natural variation of human genomes (“1000 genome project”) http://www.1000genomes.org/page.php

• Sequencing of 802 eukaryotic species: http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi

• Sequencing of extinct species: Neanderthal http://www.broadinstitute.org/

NGS, application 4: identifying genetic defects in whole genome

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June 2010; vol. 42, pp. 483-486

Schinzel-Giedion syndrome

Severe mental retardation Distinctive facial features Multiple congenital abnormalities Neoplasias Sporadic occurrence (de novo mutations?)

Schinzel-Giedion syndrome

Sequence analysis of all exons of 18,000 genes of 4 unrelated patients

Exons constitute 1% of human genome

De novo SETBP1 mutations in 12 patients with Schinzel-Giedion syndrome

Normal Normal

**Mutations: Asp868AsnGly870SerIle871Thr

Normal Normal

Normal

• 1953: Watson, J. & Crick, F.: The double helix

• 1977: Sanger, F. et al.: DNA sequencing

• 1983: Mullins, K.B.: Polymerase Chain Reaction

• 2005: Margulies, M. et al. and many others: Next generation sequencing

Milestones in molecular genetics

The impact of next generation sequencingon clinical genetics

Predictions: • in 2013 more than 90% of all human disease genes have been identified.

• in 2013 sequence analysis of all human genes will cost € 500 (Rp. 5.000.000) per person

The impact of next generation sequencingon clinical genetics

Challenges:

• to understand the effect of DNA variants

• to understand the interaction between genetic defects that can explain intra- en interfamilial variability of the expression of human disease

• to make NGS technology available to developing countries

Acknowledgments

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Kostas NikopoulosRob Collin

Ellen BloklandMarijke Zonneveld

Anneke den Hollander

Kornelia NevelingNienke WieskampMichael KwintPeer ArtsChristian GillisenAlex HoischenMichael BuckleyHans SchefferJoris Veltman