Sequence Analysis Troubleshooting Guide
iii
Table of Contents
Section I: General Diagnostic Guidelines
...............................................................................1
Raw Data - Good Raw Data Signal ...............................................................................1
Current Profiles – Normal Current Profile ....................................................................2
Section II: Troubleshooting a CEQ System Problem
............................................................4
Is it an instrument problem or a chemistry problem?....................................................4
Diagnostics using CEQ Sequencing Test Sample ..................................................4
Validation of the DTCS kit components.................................................................5
Suspect Customer Supplied Reagents.....................................................................5
Identification of Customer Supplied Template or Primer Problems ......................5
Current problems ...........................................................................................................6
Electrokinetic Injection theory................................................................................6
Diagnosing Current Problems (What does the current profile look like?) .............6
An Erratic current or “Crashed Current” ................................................................7
An Identical Erratic current in all eight Capillaries ..............................................10
Raw Signal Problems ..................................................................................................12
Diagnosing signal level problems .........................................................................12
Causes of Low Raw Data Signal ..........................................................................12
Raw Data Signal that is Too High...............................................................................21
Insertions Caused by n-1 primers .........................................................................22
Insertions Caused by Mixed Templates,
PCR Primer Carryover or Primer Mis-priming ....................................................24
High Baseline Levels ............................................................................................26
Section III: Sequencing Troubleshooting Quick Reference Table
.....................................27
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
Chemistry ....................................................................................................................33
Which primer purification method is best for sequencing primers?.....................33
How do I optimize the Preheat treatment?............................................................35
I have sufficient Raw Data signal but I didn't get a base call.
What can I do? ......................................................................................................36
What can be done to have the greatest chance of accurately
detecting heterozygotes? .............................................................................................37
Appendix A: Technical Bulletins
...........................................................................................44
