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
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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
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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
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.
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
21. Identification of a new gene for familial exudative vitreoretinopathy TSPAN12 p. Ala 237 Pro c.709 G > C
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23. NGS, application 4: identifying genetic defects in whole genome June 2010; vol. 42, pp. 483-486
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26. De novo SETBP1 mutations in 12 patients with Schinzel-Giedion syndrome Normal Normal * Normal Normal Normal * Mutations: Asp868Asn Gly870Ser Ile871Thr
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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
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
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
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
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
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
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
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