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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
[object Object],[object Object],[object Object],[object Object],Milestones in molecular genetics
Sanger sequencing: technique A T G C T T C G G C A A G A A T G C A T G C T A T G C T T A T G C T T C 1 2 5 4 3 Gene X exon exon exon exon exon PCR  amplification T A C G A A G C C G T T C T A T G DNA template C C T T T T C C A G G G A A C T T A C G A A G C C G T T C T A T G C T T C Selection on size ABI3730 3 3 3 3 3 3 Primers
Sanger sequencing: costs ,[object Object],[object Object],[object Object],Costs: € 5 per capillary = € 250 / 25.000 nt €  0.01/nt = Rp. 100/nt
Sanger sequencing: applications ,[object Object],[object Object],[object Object],[object Object],[object Object]
Genes: on average 10 exons that encode for the protein ATG TAA TGA TAG Translation stop (translation start codon for Methionine) Protein
Sanger sequencing: limitations when testing diseases with large genetic heterogeneity Disease # Genes Sanger 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
DNA-Enrichment by array sequence capture: 1.  DNA fragmentation 2.  Hybridization to synthesized probes Next generation sequencing (NGS) 5.  Sequencing  3.  Stringent washing  4.  Elution & amplification
Library preparation Emulsion-PCR Pyrosequencing NGS:  Massive parallel sequencing  (Roche 454)
NGS: 1000-fold increase in output ,[object Object],[object Object],[object Object],[object Object],[object Object],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)
Molecular Diagnostics: Sanger sequencing vs NGS Disease # Genes Sanger sequencing costs NGS costs 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
NGS, application 1: identifying defects in known disease genes Disease # Genes NGS 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 2: identifying genetic defect in genomic region Identification of a new gene for familial exudative vitreoretinopathy Nikopoulos K. et al.  Am J Hum Genet.  86 :240-247, 2010.
Familial exudative vitreoretinopathy ,[object Object],[object Object]
Familial exudative vitreoretinopathy ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Linkage at chromosome 7 7 7 LOD LOD 0.50 0.00 0.50 1.00 1.50 2.00 2.50 3.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 chr chr 7 4.00
Candidate gene analysis 126.4  Mb 109.7 Mb 340 genes
Candidate gene analysis PhyloP score:  conservation of a nucleotide on a given   sequence among 44 vertebrate species. Position reference allele Ref. allele Variant allele Total # of reads # of variant reads % variant reads Ref. amino acid Variant amino acid Gene PhyloP score 120216091 C G 20 10 50 A P TSPAN12 5.32 98870495 G A 26 16 62 R C PTCD1 3.06 100209410 G A 15 8 53 R H ZAN 1.81 99835402 C T 13 6 46 P L PILRA 1.75 113306419 C T 15 6 40 S N PPP1R3A 1.05 100473466 A G 38 13 34 T A MUC17 0.60 128099699 C G 7 5 71 I M FAM71F2 0.42 115411632 C T 14 5 36 D N TFEC -0.45
NGS of 330 genes in 40 Mb region
Identification of a new gene for familial exudative vitreoretinopathy TSPAN12  p. Ala 237 Pro c.709 G > C
NGS, application 3: sequencing of whole genomes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
NGS, application 4: identifying genetic defects in whole genome June 2010; vol. 42, pp. 483-486
Schinzel-Giedion syndrome ,[object Object],[object Object],[object Object],[object Object],[object Object]
Schinzel-Giedion syndrome ,[object Object],[object Object]
De novo SETBP1  mutations in 12 patients with Schinzel-Giedion syndrome  Normal Normal  *  Normal Normal Normal * Mutations:  Asp868Asn Gly870Ser Ile871Thr
[object Object],[object Object],[object Object],[object Object],Milestones in molecular genetics
The impact of next generation sequencing on clinical genetics ,[object Object],[object Object],[object Object],[object Object],[object Object]
The impact of next generation sequencing on clinical genetics ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Acknowledgments Kostas Nikopoulos Rob Collin Ellen Blokland Marijke Zonneveld Anneke den Hollander Kornelia Neveling Nienke Wieskamp Michael Kwint Peer Arts Christian Gillisen Alex Hoischen Michael Buckley Hans Scheffer Joris Veltman

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Next Generation Sequencing - Prof. Frans Cremers

  • 1. 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
  • 2.
  • 3. Sanger sequencing: technique A T G C T T C G G C A A G A A T G C A T G C T A T G C T T A T G C T T C 1 2 5 4 3 Gene X exon exon exon exon exon PCR amplification T A C G A A G C C G T T C T A T G DNA template C C T T T T C C A G G G A A C T T A C G A A G C C G T T C T A T G C T T C Selection on size ABI3730 3 3 3 3 3 3 Primers
  • 4.
  • 5.
  • 6. Genes: on average 10 exons that encode for the protein ATG TAA TGA TAG Translation stop (translation start codon for Methionine) Protein
  • 7. Sanger sequencing: limitations when testing diseases with large genetic heterogeneity Disease # Genes Sanger 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
  • 8. DNA-Enrichment by array sequence capture: 1. DNA fragmentation 2. Hybridization to synthesized probes Next generation sequencing (NGS) 5. Sequencing 3. Stringent washing 4. Elution & amplification
  • 9. Library preparation Emulsion-PCR Pyrosequencing NGS: Massive parallel sequencing (Roche 454)
  • 10.
  • 11. 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)
  • 12. Molecular Diagnostics: Sanger sequencing vs NGS Disease # Genes Sanger sequencing costs NGS costs 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
  • 13. NGS, application 1: identifying defects in known disease genes Disease # Genes NGS 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
  • 14. NGS, application 2: identifying genetic defect in genomic region Identification of a new gene for familial exudative vitreoretinopathy Nikopoulos K. et al. Am J Hum Genet. 86 :240-247, 2010.
  • 15.
  • 16.
  • 17. Linkage at chromosome 7 7 7 LOD LOD 0.50 0.00 0.50 1.00 1.50 2.00 2.50 3.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 chr chr 7 4.00
  • 18. Candidate gene analysis 126.4 Mb 109.7 Mb 340 genes
  • 19. Candidate gene analysis PhyloP score: conservation of a nucleotide on a given sequence among 44 vertebrate species. Position reference allele Ref. allele Variant allele Total # of reads # of variant reads % variant reads Ref. amino acid Variant amino acid Gene PhyloP score 120216091 C G 20 10 50 A P TSPAN12 5.32 98870495 G A 26 16 62 R C PTCD1 3.06 100209410 G A 15 8 53 R H ZAN 1.81 99835402 C T 13 6 46 P L PILRA 1.75 113306419 C T 15 6 40 S N PPP1R3A 1.05 100473466 A G 38 13 34 T A MUC17 0.60 128099699 C G 7 5 71 I M FAM71F2 0.42 115411632 C T 14 5 36 D N TFEC -0.45
  • 20. NGS of 330 genes in 40 Mb region
  • 21. Identification of a new gene for familial exudative vitreoretinopathy TSPAN12 p. Ala 237 Pro c.709 G > C
  • 22.
  • 23. NGS, application 4: identifying genetic defects in whole genome June 2010; vol. 42, pp. 483-486
  • 24.
  • 25.
  • 26. De novo SETBP1 mutations in 12 patients with Schinzel-Giedion syndrome  Normal Normal  *  Normal Normal Normal * Mutations: Asp868Asn Gly870Ser Ile871Thr
  • 27.
  • 28.
  • 29.
  • 30. Acknowledgments Kostas Nikopoulos Rob Collin Ellen Blokland Marijke Zonneveld Anneke den Hollander Kornelia Neveling Nienke Wieskamp Michael Kwint Peer Arts Christian Gillisen Alex Hoischen Michael Buckley Hans Scheffer Joris Veltman

Notes de l'éditeur

  1. Many monogenetic diseases have more than one possible underlying gene Complex monogenic disorders Number of genes varies a lot For some diseases no routine diagnostics available, since there are too many genes to be tested Medical need to sequence more! Limitations in Sequencing capacities + front end methods, i.e. enrichment BUT: technical limitations Definite need for novel (non-PCR based) front-end methods! Capillary (Sanger) Sequencing: 96/384-well, i.e. ~50-400 kb output/run
  2. Many monogenetic diseases have more than one possible underlying gene Complex monogenic disorders Number of genes varies a lot For some diseases no routine diagnostics available, since there are too many genes to be tested Medical need to sequence more! Limitations in Sequencing capacities + front end methods, i.e. enrichment BUT: technical limitations Definite need for novel (non-PCR based) front-end methods! Capillary (Sanger) Sequencing: 96/384-well, i.e. ~50-400 kb output/run
  3. Many monogenetic diseases have more than one possible underlying gene Complex monogenic disorders Number of genes varies a lot For some diseases no routine diagnostics available, since there are too many genes to be tested Medical need to sequence more! Limitations in Sequencing capacities + front end methods, i.e. enrichment BUT: technical limitations Definite need for novel (non-PCR based) front-end methods! Capillary (Sanger) Sequencing: 96/384-well, i.e. ~50-400 kb output/run
  4. Many monogenetic diseases have more than one possible underlying gene Complex monogenic disorders Number of genes varies a lot For some diseases no routine diagnostics available, since there are too many genes to be tested Medical need to sequence more! Limitations in Sequencing capacities + front end methods, i.e. enrichment BUT: technical limitations Definite need for novel (non-PCR based) front-end methods! Capillary (Sanger) Sequencing: 96/384-well, i.e. ~50-400 kb output/run
  5. Many monogenetic diseases have more than one possible underlying gene Complex monogenic disorders Number of genes varies a lot For some diseases no routine diagnostics available, since there are too many genes to be tested Medical need to sequence more! Limitations in Sequencing capacities + front end methods, i.e. enrichment BUT: technical limitations Definite need for novel (non-PCR based) front-end methods! Capillary (Sanger) Sequencing: 96/384-well, i.e. ~50-400 kb output/run
  6. Many monogenetic diseases have more than one possible underlying gene Complex monogenic disorders Number of genes varies a lot For some diseases no routine diagnostics available, since there are too many genes to be tested Medical need to sequence more! Limitations in Sequencing capacities + front end methods, i.e. enrichment BUT: technical limitations Definite need for novel (non-PCR based) front-end methods! Capillary (Sanger) Sequencing: 96/384-well, i.e. ~50-400 kb output/run
  7. Many monogenetic diseases have more than one possible underlying gene Complex monogenic disorders Number of genes varies a lot For some diseases no routine diagnostics available, since there are too many genes to be tested Medical need to sequence more! Limitations in Sequencing capacities + front end methods, i.e. enrichment BUT: technical limitations Definite need for novel (non-PCR based) front-end methods! Capillary (Sanger) Sequencing: 96/384-well, i.e. ~50-400 kb output/run