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

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

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

Troubleshooting Tools Sequencing Test Mix (Part ) –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

Troubleshooting Tools Sequencing Test Mix & pUC18dG/-47 –Method Seq-Test >98% accuracy (2%N) Align with pUC18dG

Sequence Trace

Abnormal Sequence Trace!

Normal Raw Data and Current LFR-1 Method

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

Current Problems Gradually decreasing current => short reads

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

Current Problems “Crashed” current –Short/failed read –Inaccurate base calling

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

Current Problems –Optimizing 96°C –1 minute –2 minutes –3 minutes 85°C –5 minutes

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

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

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

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

Normal Raw Data and Current LFR-1 Method

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

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

Low Raw Data Signal [DNA Template] Too little DNA Template –Not enough reaction products Add more DNA Correct [DNA Template] femtomoles for dsDNA femtomoles for ssDNA femtomoles for PCR products

DNA Quantity Table

Quantitation of [Template DNA] –Agarose Gel –Fluorescence Picogreen –Spectrophotometer

Molecular Weight Markers

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

Low Raw Data Signal Primer Problems Low [primer] Inefficient primer binding –Primer design –Cycling conditions Cycle number Annealing temperature

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

Primer Design Primer Interactions –Hairpins –Primer dimers

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

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

Low Raw Data Signal Dye Degradation Typically red/black dyes degrade first –Dyes stable in: Reaction buffer SLS under mineral oil –Bad formamide Use CEQ kit SLS (part ) 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

Low Raw Data Signal Dye Degradation Typically red/black dyes degrade first

Low Raw Data Signal Ethanol Contamination Typically red dye affected the most Massive noise in the D1 channel Low sample signal

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

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 Data Truncated (off- Scale)

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

Correct Raw Data Signal Analysis Poor or Fails Insertions Caused by: –Mixed Templates –PCR Primer Carryover –Primer Mis-priming

Correct Raw Data Signal Analysis Poor or Fails Template contamination

Correct Raw Data Signal Analysis 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

Low Level Contamination of PCR Template

Reduce injection Reduce amount of template Increase annealing temp in PCR reaction Re design PCR primers

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

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

Pre-peak Reduction

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

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

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

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

G/C rich templates Chemistry Modifications –DMSO DO NOT use –Single Strand Binding Protein (Stratagene) 0.5 µg per 20 µl DTCS reaction Better with DTCS than Quickstart –Betaine Final concentration 0.7 to 2M per sequencing reaction Good for both DTCS and Quickstart –DeazaGTP Replace dNTP mix in DTCS kit –Separate components from Sigma, USB etc Add dNTPs to a final concentration of 0.6mM –Use deazaGTP in place of dITP Use 72 ° C extension temperature

PolyA and PolyT Problem is enzyme slippage

PolyA and PolyT Problem is enzyme slippage –Pre-peak reduction may help Sequence specific –Redesign primer position –Try both Quickstart and DTCS chemistry

SEQUENCING Conclusion Troubleshooting Logic –“Big picture” patterns Look for problems common to: –One primer –One DNA template –One purification method –One user! –Etc….. Please ask us for help!

The pathway to Genetic Discovery begins here