CEQ Sequencing and Fragment Analysis Troubleshooting

1. CEQ Sequencing and Fragment Analysis Troubleshooting CEQ User Group Meeting Noreen Galvin Ph.D.

2. Introduction • Run Method Summary • Troubleshooting Tools • Normal traces • Current problems • Raw data problems • Mixed signals • Difficult templates

3. Run Methods • Sequencing • LFR-1 = 700bp • LFR-a = >800bp • LFR-b = 600bp • LFR-c = 500bp • Seq-Test = Sequencing Test Sample/pUC only • Fragment Analysis • Frag1 • Frag2 • Frag3 = 400bp frags • Frag4 = 600bp frags • SNP-1 = SNP anaysis • Frag-Test = Fragment Test Sample only

4. Troubleshooting Tools • Sequencing Test Mix (Part 608070) • Instrument • Gel • Capillary • Method • pUC control and -47M13 Primer • Reagents • Technique • Samples • DNA & Primer These three tests rely on the analysis of Raw Data signal, Current profiles and Analysed Data integrity

5. Troubleshooting Tools • Sequencing Test Mix & pUC18dG/-47 • Method Seq-Test • >700bp @ >98% accuracy (2%N) • Align with pUC18dG

6. Sequence Trace

7. Abnormal Sequence Trace!

8. Normal Raw Data and CurrentLFR-1 Method

9. Current Problems • Current problems are associated with the electrokinetic injection of sample and the subsequent separation of the DNA sequencing fragments in the Linear Polyacrylamide (LPA) matrix • Electrokinetic Injection • All negative charged molecules loaded • DNA sequencing products • DNA template • RNA • Protein • Salts

10. Current Problems • Gradually decreasing current => short reads

11. Current Problems • Gradually decreasing current => short reads • Some excess of supercoiled DNA template • Re-inject sample • Reduce injection • Reduce [template] • Pre-heat treatment • 96°C 1 minute

12. Current Problems • “Crashed” current • Short/failed read • Inaccurate base calling

13. Current Problems • Crashed current • Reduce [template] • Pre-heat treatment • 96°C 1 minute • Cleave template • Restriction • Remove template after reaction • Magnetic beads

14. Current Problems • Optimizing • 96°C • 1 minute • 2 minutes • 3 minutes • 85°C • 5 minutes

15. Current Problems • Identical current crash in all eight capillaries • Air bubble in manifold • Manifold purge and re-run samples

16. Current Problems • Current too high • >10µA on LFR-1 • Data starts at 20 minutes • Poor resolution • Gel degraded • Excess hours on CEQ

17. Raw Data Signal Problems • Full available range • Min Signal > 3x baseline • Max signal < 130,000 units • High Background • Reduced sensitivity with lower signals • Signal too low • Short/no read • Minimum signal:noise > 3:1 • Signal too high • Base calling errors • Too many peaks • Multiple sequencing products in one tube

18. Raw Data Background • If >6000 rfu (relative fluorescence units) • Clean capillary window • MQ water • 70% Ethanol • Blow dry

19. Normal Raw Data and CurrentLFR-1 Method

20. Low Raw Data Signal • No Signal & no dye peaks • No signal but dye peaks present

21. Low Raw Data Signal • No Signal & no dye peaks • Lost pellet • (Dye degradation) • Instrument Problem • Centrifuge • CEQ • No signal but dye peaks present • Chemistry problem • [Template DNA] • Salts in sample • [primer] • Poor primer • Polymerase inhibitor • Instrument Problem • Thermal cycler

22. Low Raw Data Signal[DNA Template] • Too little DNA Template • Not enough reaction products • Add more DNA • Correct [DNA Template] • 50-100 femtomoles for dsDNA • 25-50 femtomoles for ssDNA • 10-50 femtomoles for PCR products

23. DNA Quantity Table

24. Quantitation of [Template DNA] • Agarose Gel • Fluorescence • Picogreen • Spectrophotometer

25. Low Raw Data Signal • Salt in template or primer • Competition for injection • Add less template • Purify DNA template • Re-synthesize primer

26. Low Raw Data SignalPrimer Problems • Low [primer] • Inefficient primer binding • Primer design • Cycling conditions • Cycle number • Annealing temperature

27. Primer Design Rev (Tm 79.8) • Ideal Primer length 22-25bp 50% G/C • Tm >55°C • Annealing temp + 5°C Fwd (Tm 43.5)

28. Primer Design • Primer Interactions • Hairpins • Primer dimers

29. Primer Quality • Poor quality primer • Degraded • Freeze-thawing • Store in MQ water or 10mM Tris • Poor synthesis • Check primer integrity on CE/HPLC • Poor purification • Minimum = desalting

30. Low Raw Data SignalPolymerase Inhibitors • Polymerase inhibitors • DEPC water • Phenol • EDTA • DNA binding proteins • Other…. • Purify Template • Spin columns • Store primer • Water or Tris

31. Low Raw Data SignalDye Degradation • Typically red/black dyes degrade first • Dyes stable in: • Reaction buffer • SLS under mineral oil • Bad formamide • Use CEQ kit SLS (part 608082) • Do NOT freeze-thaw • Heating pellets to dry them • Dry in vacuum or air • No mineral oil overlay in CEQ plate • Always overlay samples in SLS

32. Low Raw Data SignalDye Degradation • Typically red/black dyes degrade first

33. Low Raw Data SignalEthanol Contamination • Typically red dye affected the most Massive noise in the D1 channel Low sample signal

34. Instrument Issues • Thermal Cycler • Heated lid • Sample evaporation with plates/caps • Block problem (PCR may still work) • Centrifuge • Refrigerated • Forces > 1000 x g for plates, >10,000 x g for tubes • CEQ • Check with sequencing test sample • Resolution and accuracy • Capillary, gel, instrument

35. Data Truncated (off-Scale) Raw Data Signal Too High • When the signal is too high this causes the data to be “truncated” (off-scale) • This can result in the addition of erroneous peaks in the base calling

36. Raw Data Signal Too High • Reduce injection of samples • Reduce [DNA template] • Reduce cycle number • Check [DNA template] before reaction

37. Correct Raw Data SignalAnalysis Poor or Fails • Insertions Caused by: • Mixed Templates • PCR Primer Carryover • Primer Mis-priming

38. Correct Raw Data SignalAnalysis Poor or Fails • Template contamination

39. Correct Raw Data SignalAnalysis Poor or Fails • Template contamination • PCR products: • Multiple products • Check on agarose • Purify from agarose • Plasmids • More than one colony • Pick another colony • Re-plate

40. Low Level Contamination of PCR Template

41. Low Level Contamination of PCR Template • Reduce injection • Reduce amount of template • Increase annealing temp in PCR reaction • Re design PCR primers

42. Correct Raw Data SignalAnalysis Poor or Fails • Primer contamination • PCR templates – primer carry-over • Purify by spin column or ExoSap • Primer degredation • N-1 peaks • Fresh primer

43. Primer Degradation • N-1 peaks • Pre-peak reduction in S/W • Resynthesize primer

44. Pre-peak Reduction

45. Pre-peak Reduction

46. Difficult Templates • Short PCR Products • Difficult to purify • G/C rich samples • Form hairpins • PolyA or polyT • Enzyme slippage

47. Short PCR Products (<200bp) • Low recovery on Spin columns

48. Short PCR Products (<200bp) • Low recovery on Spin columns • Exo-SAP purification • ExonuleaseI + Shrimp Alkaline Phosphatase • 0.05u SAP + 0.1u Exo per µl PCR reaction • 37°C for 60 minutes • Inactivate at >75°C for 15 minutes • Hold at 4°C

49. G/C rich templates

50. G/C rich templates • Range of Heat Treatments • From 1 to 5 minutes • Adjust thermal cycling conditions: • Hot start • 96 ° C for two minutes • Thirty cycles of • 96 ° C 30seconds • 60 ° C 4 minutes • Hold 4 ° C