Next Generation Sequencing Charlotte Alston NCG Mitochondrial Diagnostic Laboratory

1. Next Generation Sequencing Charlotte Alston NCG Mitochondrial Diagnostic Laboratory Newcastle upon Tyne

2. Keywords: Next Generation Sequencing Platforms Target library Sequence Capture Enhancement Bridge PCR Emulsion PCR Illumina GS FLX / 454 SOLiD Applied Biosystems

3. Introduction to different NGS platforms ►454 Life Sciences / Roche ► Illumina / Solexa ► Applied Biosystems / SOLiD ► Helicos, Polonator, Life/APG

4. Next Generation Sequencing (NGS) • Template preparation • Clonal amplification or single molecule template • Different NGS platforms • Genome alignment & assembly • Application of NGS technology in diagnostics • Future technologies

5. Template preparation • Clonal amplification • Single incorporations are below threshold of detection • Amplification achieved through emulsion PCR or solid phase amplification. • Genomic DNA is randomly fragmented • Universal adapters are added • Adapters have complementary sequences bound to fixed surface – lipid bead for emulsion PCR or a chip for solid phase amplification. • Immobilised so sequencing of molecules can be effectively multiplexed

6. Library preparation Fragment library Mate paired library

7. Advantage of mate pair library Mate paired library gives you information about location of sequences in relation to each other e.g. insert (orange) is 500bp in total; the sequenced target regions (blue) are genomically ~500bp apart. If mapped <500bp, indicates an insertion. If mapped >500bp, indicates deletion vs the reference sequence.

8. Emulsion PCR • Step 1: Ligation of universal adaptors to randomly fragmented gDNA. gDNA fragment binds to bead linker region complementary to universal adaptor (RED). • Universal adaptor allows later extension of all fragments using one PCR primer.

9. Emulsion PCR Step 2: Extension gDNA fragment is extended to generate a complementary copy attached to universal adaptor

10. Emulsion PCR Following PCR, multiple identical copies of the gDNA fragment have been created and are attached to the bead. 100-200 million beads can be bound to glass slides or each bead deposited into individual wells of a PTP (PicoTiterPlate).

11. Solid Phase amplification 1 Stage 1: Universal adapters are added to both ends of random fragment DNA

12. Solid Phase amplification 1 2 Stage 2: Single stranded fragments bind randomly to complementary adapter attached to a chip/slide

13. Solid Phase amplification Extension of single stranded gDNA fragment, and formation of bridge with a nearby complementary adapter.

14. Solid Phase amplification Produces clusters of full length gDNA fragment for sequencing

15. Solid Phase amplification

16. Next Generation Sequencing (NGS) Roche/454 GS FLX

17. Next Generation Sequencing (NGS) ► Roche GS FLX / 454 Life Sciences Uses emulsion PCR to produce 100-200 million beads with clonally amplified gDNA fragments

18. Next Generation Sequencing (NGS) ► Roche GS FLX / 454 Life Sciences Post-emulsionPCR, products are passed over picotiter plate; pores large enough for a single coated bead 1 clone/well

19. Next Generation Sequencing (NGS) ► Roche GS FLX / 454 Life Sciences Sulphurylase and luciferase enzyme coated beads (orange spheres in diagram) are added to PicoTiterPlate wells.

20. Next Generation Sequencing (NGS) ► Roche GS FLX / 454 Life Sciences Chemilumicescence corresponding to dNTP incorporation is measured by a high resolution charge coupled device (CCD) camera.

21. Next Generation Sequencing (NGS) Illumina Genome Analyzer

22. Next Generation Sequencing (NGS) ►Illumina Genome Analyzer sequencing.

23. Next Generation Sequencing (NGS) ►Illumina Genome Analyzer sequencing.

24. Next Generation Sequencing (NGS) ►Illumina Genome Analyzer sequencing. Laser excitation of next dNTP in sequence Capture of fluorophore

25. Next Generation Sequencing (NGS) ►Illumina Genome Analyzer sequencing. The computer monitors each dNTP incorporation, recording the fluorescence. Sequences are determined from clusters concurrently; high throughput of sequencing Fast and more accurate vs GS FLX, but much shorter reads of 75 and 50 bases Cheaper vs GS FLX

26. Next Generation Sequencing (NGS) SOLiD Applied Biosystems

27. SOLiD – Applied Biosystems Uses emulsion PCR to produce 100-200 million beads with clonally amplified gDNA fragments. Ligated to plate via covalent binding using target linkers and gDNA-bound target linkers.

28. SOLiD – Applied Biosystems Sequencing by ligation 8bp probe (specific at first two bp) is ligated and fluorescence is emitted and measured. Fluorophore is removed by cleavage, along with the 3 terminal nucleotides of the probe.

29. SOLiD – Applied Biosystems PROBLEM: From the fluorophore coding table, this red fluorophore could correspond to either AT, CG, GC or TA

30. SOLiD – Applied Biosystems A T SOLUTION: If you use the last nucleotide of the adapter sequence (known & universal to all target fragments) as the first to be probed, the sequence can be elucidated. e.g. last nt of adapter = T

31. SOLiD – Applied Biosystems Sequencing by ligation Next 1,2-probe is added and flourescence is measured. As before, fluor is cleaved along with 3 nucleotides of the primer.

32. Round 1 Round 2 (staggered by 1bp) 1 2 SOLiD – Applied Biosystems REPEAT using n-1 seq primer Results from two rounds of seq Round

33. SOLiD – Applied Biosystems Process is repeated five times using primer n in 1st cycle, n-1 primer in 2ndcycle (etc) until an n-4 primer is used at 5th cycle. RESULT: all nucleotides are probed in duplicate; fewer errors.

34. SOLiD – Applied Biosystems Use the nucleotide decoder chart to determine sequences of fluorophores

35. Comparison of Sanger vs NGS Platforms

36. Next Generation Sequencing approaches: • All introns and exons - the genome • All coding regions - the exome • Targeted approach - User defined genes

37. Target enrichment for whole exome sequencing Sequence capture enrichment (SCE) Random gDNA fragments are ligated to adapter molecules Hybridised with biotinylated RNA baits; bind to streptavidin coated magnetic beads; magnetic capture of target sequences (solution based capture) or Bind to complementary oligos bound to microarray chips; washing & dissociate to capture target sequences (Solid phase capture) Commercially available kits / arrays Roche/NimbleGen / Agilent/SureSelect

38. Application of NGS in diagnostics • Neonatal diabetes mellitus (NDM) • NDM is genetically heterogeneous and can be transient or persistent. • Detection of mutations in KCNJ11 or ABCC8 means diabetes can be effectively controlled by sulfonylurea; end of insulin injections for patients. • Whole exome sequencing performed on a patient with a negative screen for common NDM-associated genes, including KCNJ11 and ABCC8 • Bonnefond et al. 2010. PLoS One, Oct 26;5(10):e13630

39. Application of NGS in diagnostics • Exome sequencing for neonatal diabetes mellitus (NDM) • 187,000 coding exons using 3mg patient gDNA were captured with Agilent SureSelect HumanAll Exon kit. • Fragmentation of gDNA; fragments captured by hybridisation to the biotinylated RNA library ‘‘baits’’ • Bound genomic DNA was purified with streptavidin coated magnetic beads and re-amplified. • Whole-exome DNA library was sequenced using two channels of the Illumina Genome Analyzer IIx.

40. Application of NGS in diagnostics • Neonatal diabetes mellitus (NDM)

41. Application of NGS in diagnostics • Neonatal diabetes mellitus (NDM) • Whole exome sequencing revealed novel mutation c.1455G>C; p.Q485H in ABCC8 (dominant) • Patient treatment switched from insulin to sulfonylurea; end of injections for patients.

42. Application of NGS in diagnostics • Ocular birth defects • Genetically heterogeneous; designed custom array for SCE. 385,000 probes covering 112 candidate genes involved in coloboma, microphalmia, eye development • GS FLX was used to sequence two patient samples with known recessive PAX2 mutations; all mutations were detected with confidence, including the c.77delG (in a polyG tract). • Cost ~$8000 per two samples for 112 genes, 1017 exons • Raca et al. Mol Genet Metab. 2010 Jun;100(2):184-92

43. Application of NGS in diagnostics • Ocular birth defects c.77delG mutation • Raca et al. Mol Genet Metab. 2010 Jun;100(2):184-92

44. With great power comes…lots of SNPs Novel: not present in dbSNP130 or the eight HapMap exomes sequenced by Ng et al. 2009 Bonnefond et al. 2010. PLoS One, Oct 26;5(10):e13630

45. Future advances on the horizon… Nanopore sequencing => An exonuclease coupled to a nanopore => Captures DNA strand and sequentially releases dNTPs from strand. => Each dNTP disrupts the action potential by a given value whilst passing through the pore => Permits determination of DNA sequence http://www.nanoporetech.com/sequences

46. Future advances on the horizon… Pacific Biosciences Polymerase is anchored to the bottom of a well. Fragmented DNA sample is fed through polymerase “Real time sequencing” fluorescent signals are read as the rate of polymerase dNTP extension No need to amplify a DNA sample using the polymerase chain reaction (PCR) so minimises errors

47. Future advances on the horizon… Nanopore sequencing http://www.nanoporetech.com/sequences Alpha haemolysin nanopore showing cyclodextrin adapter molecule (the DNA binding site).

48. Future advances on the horizon… Nanopore sequencing http://www.nanoporetech.com/sequences

49. References Voelkerding KV, Dames SA, Durtschi JD. Next-generation sequencing: from basic research to diagnostics.Clin Chem. 2009 Apr;55(4):641-58 Bonnefond A, Durand E, Sand O, De Graeve F, Gallina S, Busiah K, Lobbens S, Simon A, Bellanné-Chantelot C, Létourneau L, Scharfmann R, Delplanque J, Sladek R, Polak M, Vaxillaire M, Froguel P. Molecular diagnosis of neonatal diabetes mellitus using next-generation sequencing of the whole exome. PLoS One. 2010 Oct 26;5(10):e13630. Metzker ML. Sequencing technologies - the next generation. Nat Rev Genet. 2010 Jan;11(1):31-46. Gilissen C, Arts HH, Hoischen A, Spruijt L, Mans DA, Arts P, van Lier B, Steehouwer M, van Reeuwijk J, Kant SG, Roepman R, Knoers NV, Veltman JA, Brunner HG.Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome. Am J Hum Genet. 2010 Sep 10;87(3):418-23. Raca G, Jackson C, Warman B, Bair T, Schimmenti LA. Next generation sequencing in research and diagnostics of ocular birth defects. Mol Genet Metab. 2010 Jun;100(2):184-92 Nanopore website: http://www.nanoporetech.com/sequences Website akin to SOLiD for dummies! http://arstechnica.com/science/guides/2009/12/dna-sequencing-gets-solid-with-built-in-error-detection.ars Official SOLiD information http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html