Beckman Coulter CEQ 8000 series Troubleshooting Manual
  1. Manuals
  2. Brands
  3. Beckman Coulter Manuals
  4. Measuring Instruments
  5. CEQ 8000 series
  6. Troubleshooting manual

Beckman Coulter CEQ 8000 series Troubleshooting Manual

Genetic analysis system
Hide thumbs
Table of Contents

Advertisement

Quick Links

390216-AB
July 2003
TM
CEQ
8000
™
Series
Genetic
Analysis
System
Sequence Analysis

Troubleshooting

Guide
Beckman Coulter, Inc.
4300 North Harbor Boulevard, Fullerton, CA 92835
Copyright 2003 Beckman Coulter, Inc.

Advertisement

Table of Contents
loading

Related Manuals for Beckman Coulter CEQ 8000 series

Summary of Contents for Beckman Coulter CEQ 8000 series

  • Page 1: Troubleshooting

    390216-AB July 2003 8000 ™ Series Genetic Analysis System Sequence Analysis Troubleshooting Guide Beckman Coulter, Inc. 4300 North Harbor Boulevard, Fullerton, CA 92835 Copyright 2003 Beckman Coulter, Inc.
  • Page 2 Copyright, Licenses and Trademarks  Beckman Coulter, Inc., 2003. All rights reserved. No part of this publication may be reproduced, transcribed, transmitted, or translated into any language in any form by any means without the written permission of Beckman Coulter, Inc.
  • Page 3 Section IV: Sequence Analysis FAQ Sheet................32 General Operation .......................32 Can I perform DNA sequencing and fragment analysis on the same plate using the CEQ 8000 Series System?........32 Can I switch to another database to inspect old data while new data is being collected?................32 What are the differences between the LFR-1, LFR-a, LFR-b and LFR-c methods?....................32...

  • Page 5: Section I: General Diagnostic Guidelines

    Section I: General Diagnostic Guidelines Section I: General Diagnostic Guidelines The CEQ System has many useful features that allow the user to identify and resolve problems quickly. The two most useful features in this regard are the Raw Data and the Current Profile.

  • Page 6: Current Profiles – Normal Current Profile

    Section I: General Diagnostic Guidelines Current Profiles – Normal Current Profile The CEQ System continuously monitors current in all 8 capillaries while the system is running. This current trace can be extremely useful in diagnosing certain problems. The current profile should look similar to Figure 2. The current ramps up to the final level in one stage.
  • Page 7 Section I: General Diagnostic Guidelines The software allows for the development of custom separation methods. If a significantly higher separation voltage (6–12KV) is desired, then the advanced features of assigning the separation conditions should be employed. The advanced features allow for a gradual, stepped ramp to the final separation voltage. Such a gradual voltage increase allows the linear polyacrylamide (LPA) to slowly heat up and thereby reduces possible thermal expansion out of the capillary caused by the rapid exposure to high voltages.

  • Page 8: Section Ii: Troubleshooting A Ceq System Problem

    CEQ Sequencing Test Sample. The individual components should be tested in separate experiments to identify the faulty component. If an acceptable result is still not achieved after substituting all new components call the Beckman Coulter Field Service Representative for service on the CEQ system.

  • Page 9: Validation Of The Dtcs Kit Components

    -47 sequencing primer provided in the DTCS kit. This test is designed to try to discriminate if the chemistry problems are due to the Beckman Coulter, Inc. provided reagents or customer supplied reagents. Results that pass the system specification of 98% accuracy at 700 bases (using the LFR-1) confirm the quality of the Beckman Coulter, Inc.

  • Page 10: Current Problems

    Section II: Troubleshooting a CEQ System Problem Current problems Electrokinetic Injection theory 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. The electrokinetic injection system will “load” negatively charged molecules on to the LPA matrix at the tip of the capillary.

  • Page 11: An Erratic Current Or "Crashed Current

    Section II: Troubleshooting a CEQ System Problem If sufficient raw data signal is present, reduce the amount of template present in the cycle sequencing reaction. In this case, just adding half the amount of template may eliminate the current problem (i.e. if the original template amount was 100 femtomoles (fmoles), use 50 fmoles).
  • Page 12 Section II: Troubleshooting a CEQ System Problem If the current is unstable or erratic, then the migration times of the bases will be adversely affected and this can lead to inaccuracies in the base calling. During the time that the current is not being maintained, as shown in Figure 4, the base migration will be slowed and the accuracy of base calling will be low.
  • Page 13 Once again, the preheat treatment (see CEQ DTCS Chemistry, Beckman Coulter, Inc. P/N 718119) nicks the template DNA to alleviate the supercoiling of the plasmid DNA and nicked DNA does not “clog”...

  • Page 14: An Identical Erratic Current In All Eight Capillaries

    Section II: Troubleshooting a CEQ System Problem An alternative to preheat treatment is to cleave the plasmid with a restriction endonuclease before adding the template to the sequencing reaction. In effect, this method converts a supercoiled plasmid into a linear molecule, which causes the DNA template to perform like a PCR product (no current problems observed).
  • Page 15 Do 1–2 manifold purges from the Direct Control menu on the CEQ Run application. Check the sample plate for air bubbles before loading it onto the CEQ. Call Beckman Coulter, Inc. service if the first two actions do not fix the problem. Sequence Analysis Troubleshooting Guide...

  • Page 16: Raw Signal Problems

    Section II: Troubleshooting a CEQ System Problem Raw Signal Problems Types of Raw data signal problems There are several types of raw data signal problems. The most common problem is signal levels that are too low. In this case there is insufficient fluorescence signal (shown as Activity (cnts) on the electropherogram) to generate an accurate base call.
  • Page 17 Section II: Troubleshooting a CEQ System Problem Shown, in Figure 8, is an example of a DNA sequencing sample where not enough template DNA was added. In this example less than 25 fmoles of template was added to the sequencing reaction. Notice the very low Raw Data signal throughout the run and particularly the low Raw Data signal in the red, green and black traces.
  • Page 18 Section II: Troubleshooting a CEQ System Problem If the correct amount of template was added and the preheat treatment does not yield a substantial increase in Raw Data signal increase the number of cycles in the thermal cycling program from 30 to 40 or 50. If the correct amount of template was added and the Preheat Treatment and / or increasing cycle number does not yield a substantial increase in Raw Data signal, a DNA Polymerase inhibitor may be present (do not resuspend DNA in DEPC...
  • Page 19 Section II: Troubleshooting a CEQ System Problem In the following examples (Figure 9 and Figure 10), the template is the same, but two different primers have been used. Primer 1 has a T of 43.5°C, a 31.6% GC content and is 19 bases long. Primer 2 has a T of 79.8°C, a 62.5% GC content and is 24 bases long.
  • Page 20 Section II: Troubleshooting a CEQ System Problem Corrective Actions Add the correct amount of the primer to the reaction. This will require quantitation of the primer by spectrophotometry. The recommended amount of primer is at least 40-fold molar excess in relation to the template. For sequencing of most template DNAs, 3–10 pmoles of primer is sufficient for a successful sequencing reaction.
  • Page 21 We do not recommend using water to resuspend the DNA sequencing fragments prior to loading on the CEQ. The Beckman Coulter, Inc. WellRED™ dyes are not stable in pure water solutions and will yield Raw Data signals similar to that of “Bad Formamide”...
  • Page 22 If using spin column purification methods make sure that the column materials do not contain salts (check with the spin column manufacturer for details for using their products with capillary sequencers). Use Beckman Coulter, Inc. supplied SLS for sample resuspension. CEQ™ 8000 Series Genetic Analysis System...
  • Page 23 Section II: Troubleshooting a CEQ System Problem Low Raw Data Signal Caused by Poor Quality Mineral Oil The mineral oil supplied in the DTCS kit is high quality oil containing no detectable nuclease activity. The use of other lower quality mineral oils can lead to sample degradation and hence low signal as shown below.
  • Page 24 Section II: Troubleshooting a CEQ System Problem Any one of the previously mentioned problems will yield low Raw Data signal similar to that shown in Figure 14. Figure 14: Low Raw Data signal Corrective Actions Check thermal cycler for correct performance. If you are using a robocycler optimization will be required.

  • Page 25: Raw Data Signal That Is Too High

    Section II: Troubleshooting a CEQ System Problem Raw Data Signal that is Too High In a few cases, the signal strength can be so high that it saturates the detector. This can lead to an erroneous base call where the software will artificially estimate peak height and position.

  • Page 26: Insertions Caused By N-1 Primers

    Section II: Troubleshooting a CEQ System Problem Insertions Caused by n-1 primers The presence of a significant percentage of n-1 primer in a primer preparation can lead to small peaks preceding the true sequence peaks. In certain cases these small peaks will be called as bases in the sequence.
  • Page 27 Section II: Troubleshooting a CEQ System Problem Note that the characteristic incorporation effects of thermally stable DNA Polymerase and the WellRED™ dyes can be helpful in diagnosing prepeaks from true peaks. For example; a T followed by a T always has a much larger signal for the first T than the second T (Figure 17).

  • Page 28: Insertions Caused By Mixed Templates Pcr Primer Carryover Or Primer Mis-Priming

    Section II: Troubleshooting a CEQ System Problem Insertions Caused by Mixed Templates, PCR Primer Carryover or Primer Mis-priming In many instances poor sequencing results are the cause of multiple sequencing events occurring in one sequencing reaction. These multiple sequencing events are usually caused by either mixed templates (2 or more unique templates in a reaction), PCR primer carryover (sequencing reactions primed by the PCR primer in addition to the sequencing primer) or mis-priming of the sequencing primer (the sequencing primer...
  • Page 29 Section II: Troubleshooting a CEQ System Problem Figure 18 is an example of inserted bases caused by contaminated template. The Raw Data Signal looks good but the analyzed data contains a lot of inserted bases. Should not see peaks under peaks, as the gel has one base resolving power.

  • Page 30: High Baseline Levels

    Section II: Troubleshooting a CEQ System Problem Purify the PCR products prior to using them in DTCS reactions. Use a primer that binds to a site internal to the primers used for PCR (nested primer). Select annealing temperatures that limit the annealing of mismatched primers. Unambiguous sequence regions should be chosen for selecting the priming sites.

  • Page 31: Section Iii: Sequencing Troubleshooting Quick Reference Table

    Section III: Sequencing Troubleshooting Quick Reference Table Section III: Sequencing Troubleshooting Quick Reference Table RAW DATA SIGNAL / POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS CURRENT PROBLEM PROFILE • Irreproducibility Low Raw Signal and Not enough DNA Increase the [DNA], always quantify •...
  • Page 32 Section III: Sequencing Troubleshooting Quick Reference Table RAW DATA SIGNAL / POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS CURRENT PROBLEM PROFILE Low Raw Signal and Preheat treatment on plasmid Preheat all plasmids templates Erratic or Crashed samples was not performed current profiles (plasmid/cosmids/YAC/BAC/ PAC samples).
  • Page 33 Section III: Sequencing Troubleshooting Quick Reference Table RAW DATA SIGNAL / POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS CURRENT PROBLEM PROFILE Only signal of the Failed Enzymatic Reaction Check T of primer and work out the Unincorporated correct annealing temperature dyes observed with a normal Current Profile Incorrect primer...
  • Page 34 Section III: Sequencing Troubleshooting Quick Reference Table RAW DATA SIGNAL / POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS CURRENT PROBLEM PROFILE • Irreproducibility Jump in the Raw Some sort of carry over in the Elute DNA template in sterile water or •...
  • Page 35 Section III: Sequencing Troubleshooting Quick Reference Table RAW DATA SIGNAL / POTENTIAL SOURCE OF PROBLEM POSSIBLE SOLUTIONS CURRENT PROBLEM PROFILE • Poor Resolution Check the accuracy Check pUC18 Control template—if of base calling with this is okay then the DNA template the pUC18 Control and/or primer is at fault Template.

  • Page 36: Section Iv: Sequence Analysis Faq Sheet

    CEQ 8000 Series System? If you have purchased or have upgraded to the CEQ 8000 Series System it is possible and practical to run both sequencing and fragment analysis on the same plate. The CEQ 8000 Series System upgrade uses the same 33cm capillaries and plenum for both sequencing and fragment analysis.

  • Page 37: Chemistry

    Section IV: Sequence Analysis FAQ Sheet Chemistry Which primer purification method is best for sequencing primers? The major factor with a primer that affects DNA sequence quality is the actual synthesis quality. If a synthesized primer contains little n-1, then high quality sequence can be obtained.
  • Page 38 Section IV: Sequence Analysis FAQ Sheet Figure 21: Reverse Phase HPLC Purified Primer Figure 22: Gel Filtration Primer CEQ™ 8000 Series Genetic Analysis System...

  • Page 39: How Do I Optimize The Preheat Treatment

    Section IV: Sequence Analysis FAQ Sheet How do I optimize the Preheat treatment? Shown below are some hints for optimizing the preheat treatment of plasmid DNAs. Untreated Sample (>4kb) Need to perform the preheat treatment on the DNA and WATER – Good initial starting conditions: 96°C FOR 1 MIN.

  • Page 40: I Have Sufficient Raw Data Signal But I Didn't Get A Base Call What Can I Do

    Section IV: Sequence Analysis FAQ Sheet I have sufficient Raw Data signal but I didn't get a base call. What can I do? In certain cases, the base calling algorithm cannot find the start of data. If this happens, a default start time is automatically used. The default time is determined by a combination of the separation method parameters (voltage, temperature, and voltage ramping profile) and the length (33cm or 53cm) of the capillary used to collect the data.

  • Page 41: Detecting Heterozygotes

    Section IV: Sequence Analysis FAQ Sheet What can be done to have the greatest chance of accurately detecting heterozygotes? While the default settings work well for most good sequencing results, adjustment of the parameters may be required under the following circumstances: •...
  • Page 42 Section IV: Sequence Analysis FAQ Sheet Figure 24: Too much signal can lead to bad color correction, where residual signal in the other channels has not been completely removed, leading to the insertion of peaks due to the poor color correction. Notice that analyzed peaks from Raw Data that is off-scale has slightly more rounded tips than normal peaks.
  • Page 43 Section IV: Sequence Analysis FAQ Sheet Figure 25: Raw signal strength is fine in this electropherogram (scaled to 50,000 counts). However, the presence of numerous smaller underlying peaks at many positions in the analyzed data generates an abundance of false heterozygotes.
  • Page 44 Section IV: Sequence Analysis FAQ Sheet Figure 26: Using the fast method LFR-b, there is not enough resolution to discern the G shoulder, which is heterozygous with the C at position 528. There is no setting in the heterozygote detection software that can identify this heterozygosity. CEQ™...
  • Page 45 Section IV: Sequence Analysis FAQ Sheet Figure 27: The same heterozygosity run under the same separation method as in Figure 26, LFR-b, using a primer that is closer to the heterozygosity. The resolution is slightly better, and is picked up with settings of 50/20/0.45. Sequence Analysis Troubleshooting Guide...
  • Page 46 Section IV: Sequence Analysis FAQ Sheet Figure 28: The same heterozygosity as in Figure 26 and Figure 27, using a primer that is still closer to the heterozygosity, placing the position of interest at base 227. The resolution is better than either of the two previous cases, and is picked up with settings of 50/20/0.45.
  • Page 47 Section IV: Sequence Analysis FAQ Sheet Figure 29: The slower method, LFR-a, was used to increase the resolution at 530 bases. Now settings of 50/20/0.6 identify the heterozygosity correctly. Sequence Analysis Troubleshooting Guide...

  • Page 48: Appendix A: Technical Bulletins

    Publication A-1872A Improved Sequencing of Plasmids on the CEQ 2000 by a Simple Template Pre-heating Procedure. Published by Beckman Coulter and available on the Beckman Coulter web site. Publication A-1726 High Sensitivity Analysis of Nucleotides Using Electrokinetic Injection and Sample Stacking with MECC Published by Beckman Coulter and available on the Beckman Coulter web site.
  • Page 49 Appendix A: Technical Bulletins Sequence Analysis Troubleshooting Guide...
  • Page 50 Appendix A: Technical Bulletins CEQ™ 8000 Series Genetic Analysis System...

Table of Contents