Next Generation Sequencing - Prof. Frans Cremers
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Transcript of Next Generation Sequencing - Prof. Frans Cremers
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
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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