1. Early experiences in amplicon sequencing using the Roche GS-FLX massively parallel DNA sequencer and its application within a diagnostic laboratory Louise Stanley Northern Genetics Service Newcastle Upon Tyne

2. GS-FLX - Roche Next Generation DNA Sequencer

3. GS-FLX - Roche Next Generation DNA Sequencer Based upon pyrosequencing

4. GS-FLX - Roche Next Generation DNA Sequencer Based upon pyrosequencing Sequencing ReagentsCamera

5. Work-flow Library preparation Emulsion PCR – Clonal Amplification Sequencing Data analysis

6. Library Preparation PCR amplification Purification of products Quantification Dilution to 2 x 10 5 molecules/  l Alternative methods – e.g. array based pull down by NimbleGen TM

7. Emulsion PCR + BindingEmulsion Formation PCR Amplification

8. Emulsion PCR + BindingEmulsion Formation PCR Amplification

9. Emulsion PCR + BindingEmulsion Formation PCR Amplification

10. Emulsion PCR + BindingEmulsion Formation PCR Amplification

11. Emulsion PCR + BindingEmulsion Formation Emulsion Breaking and Bead Enrichment

12. Sequencing Occurs on PicoTitre Plates Capacity of LR70 ~ 100 MB Split into 2, 4, 8 or 16 regions

13. Sequencing G T C A 1 2 3 4 5

14. G T C A G 1 2 3 4 5 G

15. G T C A G T 1 2 3 4 5

16. Sequencing G T C A G T C 1 2 3 4 5 G T

17. Sequencing G T C A G T C A 1 2 3 4 5 G T A A

18. Sequencing G T C A G T C A G 1 2 3 4 5 G T A A G

19. Sequencing G T C A G T C A G T 1 2 3 4 5 G T A A G T T T T

20. Sequencing G T C A G T C A G T C 1 2 3 4 5 G T A A G T T T T C C C

21. Data Analysis Sequencing reads aligned to reference sequence Software searches for variants – presented in both tabular and graphical formats Some manual inspection of sequence data recommended

22. Data Interpretation Length of amplicon sequence Number of reads across amplicon Variants Variant Table Consensus Global Alignment Plot

23. Data Interpretation Variant Table Consensus Global Alignment Plot T>CC>T

24. Mutation Detection ReferenceExonMutationMale/Female D853563c.106_109delinsTM D995199c.833C>TF D9952031c.4290_4291delM D9952136c.5067_5068delCCinsGF D9952258c.8668+1G>AM D4268766c.9568C>TM D9952369c.10086+1G>TF 7 patients with different mutations in dystrophin gene examined

25. Mutation Detection All mutations identified and called by software in variant tables Investigated level of coverage required for mutation detection Achieved by mixing patient libraries with mutations with WT library [c.4290_4291del; male][c.833C>T; female]

26. Mutation Detection Number of Regions Reads per Region Mb per Region Amplicons per Region 2200 00050~ 3300 470 00017.5~ 1150 830 0007.5~ 500 169 – 12 0002.25 – 3.0~ 150 - 200 Dilution experiments with mutation positive and wild-type samples showed ~ 60 fold coverage is required for mutation detection (including indels)

27. NimbleGen Capture Array Long oligonucleotide bound to chip Exon 2Exon 1Exon 4Exon 3 Exon 5 Fragment DNA and hybridise to NimbleGen capture array Captured DNA fragments Elute DNA and sequence using FLX

28. NimbleGen Capture Array Up to 5 MB of sequence can be captured 16 genes involved in muscular dystrophies defined – total sequence ~ 250, 000 base pairs Projected capacity – 4 patients per LR70 sequenced for 16 genes

29. NimbleGen Capture Array GeneChromosomal LocationTotal SizeCaptured sequence (to include exons) LMNA1q21.2-q21.325,4108,015 FKRP19q13.3212,4385,300 CAPN315q15.1-q21.164,21431,600 CAV33p2513,1982,347 VCP9p13.323,45212,200 SGCA17q219,9266,400 SGCB4q1217,5747,600 SGCD5q33-q34439,27815,000 SGCG13q12144,2135,250 DYSF2p13.3-p13.1233,13063,300 LDB3 (ZASP)10q22.3-q23.267,45213,400 MYOT5q3119,9799,300 DES2q358,3608,900 CRYAB11q22.3-q23.13,1443,700 FLNC7q32-q3528,85325,300 DYSTROPHINXp212,092,32829,647 TOTAL size (bp)3,202,949247,259 Capture 3 MB; capacity plate ~ 100 MB max coverage = 33 fold (100/3) 1 patient per plate Capture 250 kB; capacity plate ~ 100 MB max coverage = 400 fold process more than 1 patient per plate

30. NimbleGen Capture Array Processed one sample on LR70 PTP

31. NimbleGen Capture Array 113 MB sequence - ~ 50 % mapped to targets

32. Costs Capture Array cost ~ £1000 Sequencing ~ £2500 (LR70 PTP) Cost for 16 genes = ~ £3500 processing 1 patient per plate Conventional Sequencing = ~ £2500 for 1 patient Reduce costs by “tagging” patients prior to processing on NimbleGen capture array Application for disorders with overlapping phenotypes and multiple candidate genes (e.g. heart disease)

33. Acknowledgements Dr Jonathan Coxhead Dr Ann Curtis Dr Emma Ashton (Guy’s Hospital, London)