Next generation sequencing (NGS) provides a 1000-fold increase in sequencing output and a 100-fold reduction in cost compared to Sanger sequencing. This allows for large-scale sequencing of genes and genomes to identify genetic defects underlying human diseases. The document describes applications of NGS in clinical genetics such as sequencing known disease genes, identifying new disease genes, sequencing whole genomes, and sequencing all human genes in patients. It also discusses future predictions and challenges regarding use of NGS in clinical genetics.

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. 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

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. 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

5. 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

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. 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

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. Familial exudative vitreoretinopathy Fundus: avascular zone

16. Familial exudative vitreoretinopathy Fundus: avascular zone retinal detachment “ stretched/dragged” vasculature Visual acuity: normal  blindness

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. NGS, application 3: sequencing of whole genomes 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/

23. NGS, application 4: identifying genetic defects in whole genome June 2010; vol. 42, pp. 483-486

24. Schinzel-Giedion syndrome Severe mental retardation Distinctive facial features Multiple congenital abnormalities Neoplasias Sporadic occurrence ( de novo mutations?)

25. Schinzel-Giedion syndrome Sequence analysis of all exons of 18,000 genes of 4 unrelated patients Exons constitute 1% of human genome

26. De novo SETBP1 mutations in 12 patients with Schinzel-Giedion syndrome  Normal Normal  *  Normal Normal Normal * Mutations: Asp868Asn Gly870Ser Ile871Thr

27. 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

28. The impact of next generation sequencing on 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

29. The impact of next generation sequencing on 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

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