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Section I: General Diagnostic Guidelines

Sequence Analysis Troubleshooting Guide

1

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

. In certain cases, the analyzed data can be used, but

only after

the

raw data and current profile have been shown to be good.

Raw Data - Good Raw Data Signal

The raw data generated by the CEQ System is displayed during the actual run and is
also accessible to the user in the “Analysis Application.” The major diagnostic to use
when looking at raw data is the signal strength. In general, signal strength will
gradually decline as a run progresses as shown below:

In the example shown in Figure 1, with a start time of 20 minutes for the LFR-1
method, the signal strength at the beginning of the run is approximately 70,000 counts,
decreasing to about 10,000 counts at the end of the run. Signal strength is important
because without sufficient signal, it is very unlikely that accurate base calls can be
made. The above example shows excellent signal strength; however, it is not necessary
for each sample to have such high signal in order to obtain accurate base calls. The
important number to remember is signal to noise ratio. With a signal to noise ratio of
about 3:1, the software should give an accurate base call. For example, a profile with a
starting signal strength of 25,000 counts and an ending signal strength of 5,000
(assuming a background of about 1,500) should still give an accurate base call.

Figure 1:

Good Raw Data Signal

Profile starts high, gradually decreases in
signal strength as the run progresses

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Содержание CEQ 8000 series

Страница 1: ...M 390216 AB July 2003 Beckman Coulter Inc 4300 North Harbor Boulevard Fullerton CA 92835 Copyright 2003 Beckman Coulter Inc CEQ 8000 Series Genetic Analysis System Sequence Analysis Troubleshooting Guide ...

Страница 2: ...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 The software is copyrighted and may not be altered or given to a third party without the written authorization from Beckman Coulter ...

Страница 3: ...oblems 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 ...

Страница 4: ...iv ...

Страница 5: ...wn below In the example shown in Figure 1 with a start time of 20 minutes for the LFR 1 method the signal strength at the beginning of the run is approximately 70 000 counts decreasing to about 10 000 counts at the end of the run Signal strength is important because without sufficient signal it is very unlikely that accurate base calls can be made The above example shows excellent signal strength ...

Страница 6: ...ul in diagnosing certain problems The current profile should look similar to Figure 2 The current ramps up to the final level in one stage The final current level should be approximately 5 9µA It is determined by the separation voltage which is set in the method and is maintained throughout the run Figure 2 Typical LFR 1 Current Profile No current ramp up to the final separation level in three sta...

Страница 7: ...vanced 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 An example of such a three step ramp is given in Figure 3 Figure 3 Example of a three step ramp Current ramps up to th...

Страница 8: ...uce data that has a beginning fluorescence of approximately 50 000 120 000 units and then declines gradually until the end of the run viewed in the Raw Data view of the Sequencing Analysis application In addition the current profiles should also be normal see section Current Profiles Normal Current Profile on page 2 Finally the actual base calls are checked to see if the instrument passes the syst...

Страница 9: ...der lots of DTCS kits customer supplied formamide may also be an issue The usual symptoms for poor quality sodium acetate EDTA ethanol or formamide are low raw data signal and occasionally erratic current profiles Using sodium acetate and EDTA solutions supplied by Sigma as well as the Sample Loading Solution SLS supplied in the DTCS kits will help eliminate problems caused by poor quality reagent...

Страница 10: ...rcoiled DNA molecules load on to the LPA in the tip of the capillaries they become plugged and do not allow the free flow of electrical current through the capillary Plugged capillaries exhibit two different current problems In the less severe case where the current profile shows a gradual decline without any erratic fluctuations in the current short reads are observed These short reads are due to...

Страница 11: ...mercial DNA preparation methods yield template DNA that is essentially free of contaminating macromolecules 6 Remove the template from the sequencing reaction This is a very elaborate process requiring special primers and reagents but also yields sequencing reactions that produce no current problems The most common technique is to use a biotinylated sequencing primer and the DynaPureII sequencing ...

Страница 12: ...he base call the total read length will also be diminished simply due to the fact that the migration of the bases has been slowed from the normal run conditions In other cases current failures can be more significant Figure 5 shows a current profile that fails to reach the final running current after the normal ramping Note that the current never reaches the normal running current of 5 9 µAmps and...

Страница 13: ...quencing reaction is an obvious first step In this case adding half the amount of template may eliminate the current problem i e if the original template amount was 100 fmoles use 50 fmoles 2 Use of the preheat treatment of the template DNA prior to the cycle sequencing reaction is highly recommended for supercoiled plasmid samples and is required to solve erratic current problems or current crash...

Страница 14: ...reagents but also yields sequencing reactions that produce no current problems The most common technique is to use a biotinylated sequencing primer and the DynaPureII sequencing reaction cleanup kit Dynal Inc Oslo Norway product number 603 05 This purifies the sequencing reaction products from contaminants such as the template DNA unincorporated dye terminators dNTPs and other salts An Identical E...

Страница 15: ...n the manifold side of the CEQ gel system Corrective actions to solve manifold bubble current problems 1 Do 1 2 manifold purges from the Direct Control menu on the CEQ Run application 2 Check the sample plate for air bubbles before loading it onto the CEQ 3 Call Beckman Coulter Inc service if the first two actions do not fix the problem Figure 7 Manifold bubble current problem ...

Страница 16: ...cation The data which is plotted as Activity cnts versus Time Minutes should show fluorescent signal Activity cnts in the range of 5 000 125 000 cnts The signal will be high in the beginning and gradually decline throughout the run Causes of Low Raw Data Signal Low Raw Data Signal due to not enough template Low raw data signal can be caused by a variety of issues One of the most common causes is l...

Страница 17: ...uire quantitation of the template DNA by spectrophotometry in the case of commercial DNA preparations or by estimation using agarose gel electrophoresis and comparison to a know quantity of DNA 2 Alternatively the user could try a dilution series with the same template starting with an amount that is obviously too high and ending with an amount which is much too low This method assumes that the us...

Страница 18: ...ata signal Many suppliers of oligonucleotides provide sufficiently pure preparations of primers Most of these vendors will provide desalted primers which function well for DNA sequencing However further purification of the primers by TSP Trityl Specific Purification or HPLC High Performance Liquid Chromatography is highly recommended when the highest quality DNA sequencing results are desired Also...

Страница 19: ...t two different primers have been used Primer 1 has a Tm of 43 5 C a 31 6 GC content and is 19 bases long Primer 2 has a Tm of 79 8 C a 62 5 GC content and is 24 bases long The effect on the data obtained from the CEQ is clear With a less than efficient reaction the signal level is low and the base calling accuracy is not optimal Figure 9 Primer 1 Figure 10 Primer 2 ...

Страница 20: ...ration is pure Using reputable oligonucleotide vendors is your best way to get high quality primers If possible have the primers purified by TSP or HPLC 3 Always design the sequencing primers for use in Cycle sequencing reactions This means designing primers with high Tm s and no possibility of forming secondary structures or primer dimers There are many DNA analysis programs available for checkin...

Страница 21: ...le was less than 90 40 errors at 500 bases Corrective actions 1 Use the Beckman Coulter Inc supplied Sample Loading Solution SLS 2 Do not freeze thaw the SLS or formamide solutions Store aliquots at 20 C in a non frost free freezer and use the aliquots only once Low Raw Data Signal Due to the Use of Water as the Sample Loading Solution We do not recommend using water to resuspend the DNA sequencin...

Страница 22: ... the fluorescently labeled DNA molecules The sources of the excess salts are improperly purified sequencing reactions and decomposed formamide Corrective actions 1 Follow the ethanol precipitation procedure in the CEQ Dye Terminator Cycle Sequencing protocol Beckman Coulter Inc P N 608019 2 If using spin column purification methods make sure that the column materials do not contain salts check wit...

Страница 23: ...Raw Data Signal Caused by Thermal Cycling Problems The CEQ DTCS chemistry uses thermal cycling to produce a linear amplification process called cycle sequencing to generate sufficient fluorescent signal to generate an accurate sequencing base call The Thermal Cycler program and performance are critical to achieve optimal results There are several possible problems that are associated with the ther...

Страница 24: ...erature ramp is set to maximum 3 Check primer melting temperature if the annealing temperature is too high for the primer you will need to lower it 4 Make sure you are using PCR tubes or plates The CEQ sample plate has been proven to work on the MJ PTC 200 and P E 9600 thermal cyclers 5 Make sure strip caps A sealing film or sealing mat are put on the PCR plate correctly The lack of a tight seal w...

Страница 25: ...nts and looking at the peak shapes the user can determine if peaks are over ranged If the peaks are squared off at the top see the blue peaks in Figure 15 then the detector is saturated and the peaks are over ranged Corrective Action 1 If the peaks are too high the simplest solution is to rerun the same sample using a shorter injection time for example 7 5 seconds instead of 15 seconds 2 Use less ...

Страница 26: ... be called as bases in the sequence This will most often occur in a sequence that has a G followed by an A However multiplets i e TT or CC may also yield insertions In Figure 16 the presence of a large amount of n 1 primer 20 in the sequencing primer causes each true sequencing peak to be preceded by a smaller prepeak As can be seen in Figure 16 in many cases these prepeaks are called as bases Fig...

Страница 27: ...otide purification methods that rely on chromatography can not completely separate the n 1 from full length oligonucleotides and therefore can not rescue a contaminated primer preparation 2 If you synthesize sequencing primers using an automated DNA synthesizer use the High quality settings on your instrument This usually involves using increased amount of capping reagents and chemical coupling ti...

Страница 28: ...ain a mixed template from PCR samples cut from gels e g differential display or from PCR amplified template that contain more than one amplified fragment It is also seen with plasmid samples when for example more than one recombinant colony is inoculated into a single culture In this case the initial analyzed data is good common vector sequence but the inserted region is different and will generat...

Страница 29: ...ixed templates 2 In some instances when the contaminating templates are present in very small quantities simple dilution of the sample will decrease the contaminating sequence to a level where an acceptable sequence call can be made Figure 18 Insertions Caused by Multiple Sequencing Events The Raw Data Signal looks good but the analyzed data contains a lot of inserted bases Should not see peaks un...

Страница 30: ...mance of the CEQ System is achieved when the baselines for the 4 channels blue green black and red are below 6 000 cnts Shown in Figure 19 are two examples of dirty capillaries The example on the left may yield acceptable quality data The example on the right shows an extreme case of a dirty capillary Corrective Action 1 Remove the capillary array at the manifold end and clean the capillary window...

Страница 31: ...mal Cycler Check that the correct method was chosen on the thermal Cycler Bad Formamide Use supplied SLS for sample resuspension Incorrect primer concentration Check Primer Concentration Incomplete resuspension of pellet in formamide Make sure that the pellet is properly resuspended in formamide or If the pUC18 Control Template does not meet the specifications Problem in post reaction cleanup of s...

Страница 32: ...ormamide etc Check method of DNA purification Incorrect method of template purification e g CsCl Phenol Chloroform or insufficient removal of Ethanol during template preparation etc Refer to the CEQ 2000 DTCS chemistry Protocol 718119 AB pg 18 and Application Note A 1872A for detail on the preheat treatment procedure Use supplied SLS for sample resuspension No signal associated with normal Current...

Страница 33: ... Identical Erratic or Crashed Current profile in All eight capillaries of the array Air bubbles coming from the Manifold end Do 2 manual Manifold Purges and 2 manual Gel Fills from the Direct Control section of the Run module and rerun the samples Note use a fresh buffer plate Manifold area dirty thus not allowing for the manifold end of the array to be sealed correctly Clean Manifold area of inst...

Страница 34: ...e breaks down into formic acid and ammonia and these ionic products compete significantly with the larger DNA ions for injection and thus cause reduction in signal intensity In addition they can also cause degradation of the sample Use SLS for sample resuspension Store and use the SLS solution correctly Primer Artifacts The analyzed data contains a lot of inserted bases Peaks under peaks in the ra...

Страница 35: ... plate Check the life of the capillaries Check life of gel Capillaries Resolution is bad Less than 6 peaks per minute Spurious peaks Late start of data even though current is normal The capillary has performed more than 100 runs The capillary coating has been damaged The tips were not kept moist The array was not stored correctly Some type of chemical was used to load the sample that destroyed the...

Страница 36: ...rent projects within the same database can be examined freely even while new data is being collected What are the differences between the LFR 1 LFR a LFR b and LFR c methods You can examine all the parameters of each separation method by clicking the Method tab in the CEQ Sample Setup module The different methods are designed for different read lengths and time constraints LFR 1 is the standard Lo...

Страница 37: ...ed Using other primer purification methods do not affect the quality of sequence as illustrated below Shown below is data generated from a primer that was purified by three different methods 1 OPC column 2 Reverse Phase HPLC and 3 gel filtration In each case because the synthesis quality was high the sequencing quality was high and the purification method had little or no effect on the results The...

Страница 38: ...Section IV Sequence Analysis FAQ Sheet 34 CEQ 8000 Series Genetic Analysis System Figure 21 Reverse Phase HPLC Purified Primer Figure 22 Gel Filtration Primer ...

Страница 39: ... Dropping Untreated Sample 4kb Need to perform the preheat treatment on the DNA and WATER Good initial starting conditions 96 C FOR 1 MIN If current is still dropping the treatment has to be optimized further 96 C FOR 3 MIN If the raw signal declines rapidly after either of the above treatments change the heating conditions to 86 C FOR 5 MIN DO NOT PREHEAT the pUC18 Control Template Late start of ...

Страница 40: ...metimes simply by setting the start of data manually samples can be analyzed accurately To manually set the start of data expand the raw data near where the initial peaks are seen Examine the data and look for a peak that is DNA i e not unincorporated dye terminators which will usually be the first eluted peaks and be very large and broader than the DNA peaks and has a sharp profile Reanalyze the ...

Страница 41: ...te in the run it is recommended that the separation method LFR a be used Other than this recommendation there is no way for the user to know how aggressive to be with the heterozygote detection settings on his own data without experimentation On clean data false heterozygosities may be absent with Average peak spacing of 90 and Sensitivity settings as high as 0 75 However routinely using these set...

Страница 42: ... 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 The Raw Data is scaled from 0 to 130 000 counts ...

Страница 43: ...s Troubleshooting Guide 39 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 ...

Страница 44: ...netic Analysis System 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 ...

Страница 45: ...ysis Troubleshooting Guide 41 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 ...

Страница 46: ...stem 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 ...

Страница 47: ...quence Analysis FAQ Sheet Sequence Analysis Troubleshooting Guide 43 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 ...

Страница 48: ...ulter and available on the Beckman Coulter web site Publication A 1774A Quantitative Capillary Electrophoretic Analysis of PCR Products Published and available at Published by Beckman Coulter and available on the Beckman Coulter web site Publication A 1892B Detecting Heterozygotes with the CEQ 2000XL Published by Beckman Coulter and available on the Beckman Coulter web site Publication T 1852A An ...

Страница 49: ...Appendix A Technical Bulletins Sequence Analysis Troubleshooting Guide 45 ...

Страница 50: ...Appendix A Technical Bulletins 46 CEQ 8000 Series Genetic Analysis System ...

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