Fragment Analysis Support—Troubleshooting

For data run on capillary electrophoresis platforms, the troubleshooting process consists of the following steps:

• Identify the problem, i.e., low signal intensity, broad peaks, noisy data etc. 
• Check for proper storage of reagents and expiry dates.
• Run the controls. For fragment analysis applications, running the installation standard or size standard on its own can help direct troubleshooting efforts. A possible troubleshooting procedure would be:

1. Mix 12.5 μL of HiDi™ Formamide and 0.5 μL of Internal Size Standard (i.e., LIZ™ 600 Dye Size Standard, ROX™ 500 Dye Size Standard) per well/capillary (for example, A 16 capillary array would need 16 wells, each containing 12.5 μL HiDi™ Formamide and 0.5 μL of Internal Size Standard). 

2. Run the standards using Standard Run Modules. Sizing should pass in the Analysis Software using a Default Analysis Method and Size Standard Definition.

This can be used to determine if the size standards, instrument hardware, and consumables on the instrument (i.e., polymer, buffer, capillary/array) are working properly and if the proper software settings have been selected for the analysis. If the standards do not look good and weekly maintenance has not been performed, perform the weekly maintenance and re-run the standards. If the standards still do not look good, contact Technical Support at techsupport@thermofisher.com. If the installation/size standard plate looks good, go to step 3:

3. Set up a plate of PCR reactions using a laboratory internal DNA control sample or your QC DNA, as per your laboratory’s established protocol.

4. After the PCR reaction is complete, dilute the sample as per your laboratory protocol and mix 1 μL of diluted sample, 0.5 μL Internal Size Standard, and 10.5 μL HiDi™ Formamide. Alternatively, the diluted sample can be added to the size standard-only plate prepared in step 1, to save on reagents.

5. Denature the sample at 95 degrees C for 3 minutes, and place on ice for 3 minutes. 

6. Run the samples using Standard Run Modules. 

This can help determine if the problem is with the chemistry, thermal cycler, template, or the primers. Running the samples on an agarose gel can sometimes help in further defining areas to troubleshoot. If the standards and internal controls all look good but you experience data quality issues with your samples, the problem is most likely in the template or primers being used for that sample. Please contact Technical Support at techsupport@thermofisher.com for further assistance.

HiDi™ Formamide acts as a denaturant and provides sample stability for the heat denaturation step and while on the instrument. DI water is not recommended for injecting fragment analysis samples as it may cause variable injection quality, variable migration of the sample, and is also prone to evaporation.

In most cases, it is not necessary to purify PCR products prior to injecting on the genetic analyzer. However, there may be instances where the salt or primer concentration of the sample is impacting signal intensity and causing poor injection, in which case, PCR purification would be recommended. 

If a multiplex PCR reaction is being performed, and one PCR product is showing low signal, it may be necessary to increase the primer-pair concentration for that target or consider amplifying separately any primer pair that fails to amplify after its concentration is increased. The singleplex PCR product can then be pooled with the multiplex PCR products prior to loading on the instrument.

A multiplex PCR involves amplifying multiple targets within the same PCR reaction whereas, in pooling PCR products, the PCR reactions are performed individually for each target (singleplex reaction) and the individual PCR products are then pooled prior to loading on the capillary electrophoresis (CE) instrument. We recommend pooling the singleplex PCR products as this allows more flexibility in obtaining signal uniformity on the instrument. A multiplex PCR can require a significant amount of optimization as there may be loss of specificity, low signal, and primer–oligomer formation. 

If the peaks in other colors are directly below the peak of interest, the issue could be that the fluorescent dye being used is not part of the selected dye set, the spectral calibration needs to be performed, or the peaks are offscale. Confirm that the dye set selected on the instrument is compatible with the dye being used, run a new spectral calibration if the correct dye set has been selected and, if the signal intensity is too high, decrease sample concentration during PCR or when preparing samples for electrophoresis. 

The dye sets, corresponding matrix standards, and corresponding dyes are shown below:

If the PCR products can be visualized on an agarose gel but not on the CE instrument, and the size standard is present in the sample file, the issue may be with the fluorescently labeled primer. Re-synthesizing the primer would be recommended. 

The signal before the 50 bp fragment is most likely excess fluorescently labeled primers or primer dimers. The extraneous peaks may not interfere with the allele calls if the PCR products are above 100 bp.

If the raw data does not show any peaks for sample or size standard, this may have multiple causes:

• A blocked capillary. Run a size standard-only sample to confirm if this is the case. 
• A bubble in the capillary may cause delayed migration. A re-injection of the sample can sometimes resolve the lack of data. 
• Air bubble at bottom of sample well. Centrifuge the plate before running.
• Autosampler calibration may be necessary.
• Degraded HiDi™ Formamide. Use properly stored HiDi™ Formamide.
• High salt concentration. Salt preferentially injects smaller fragments and inhibits injection of larger fragments, so the majority of salt may have been injected in the first injection. Re-inject the sample.

Fluorescent dyes can differ in their emission spectrum, excitation spectrum, and excitation efficiency and, as a result, the intensity of the emitted fluorescence is different for each dye and, therefore, optimization of the sample concentration needs to be performed to account for the differences in the dye signal strength. For instance, in the G5 dye set, the signal intensities from highest to lowest are 6-FAM™, VIC™, NED™, and then PET™. If the same primer sequences are labeled with 6-FAM™ and NED™, more of the NED™-labeled sample must be loaded on the instrument to have signal comparable to the same 6-FAM™-labeled sample. For a list of relative signal intensities, please see the Dyes section in the DNA Fragment Analysis by Capillary Electrophoresis Guide.

If the PCR products are run on an agarose gel and there are no bands, see here for possible reasons. If the PCR products can be visualized on an agarose gel but not on the capillary electrophoresis (CE) instrument, and the size standard is present in the sample file, the issue may be with the fluorescently-labeled primer. Re-synthesizing the primer would be recommended. 

Offscale data, or when signal intensity is too high, is a result of loading too much sample into the capillary, causing saturation of the CCD camera. The sample concentration should be lowered in one of the following ways:

• Too much template in the reaction. The template can be reduced and the PCR reaction redone.
• Decrease the amount of sample injected by further diluting the PCR product. For instance, if doing a 1:2 dilution, try 1:4, 1:5, or higher dilution of the PCR product. 
• The same plate can be re-injected, but decrease the injection time in the instrument run module. 

Flat peaks are typically an indication of data with too high of a signal, also known as offscale data. In addition to flat peaks, the sample may show pull-up peaks directly under the offscale peaks. The sample concentration should be reduced in one of the following ways:

• Decrease the amount of sample injected by further diluting the PCR product. For instance, if doing a 1:2 dilution, try 1:4, 1:5, or higher dilution of the PCR product. 
• The same plate can be re-injected, but decrease the injection time in the instrument run module. 

Here are possible causes and solutions:

• Broadening of sample peaks can start to occur if the polymer or buffer has expired or is degraded. 
• Capillary array degradation and sample degradation can also cause broader peaks. If the samples have been on the instrument for more than 24 hrs, prepare a fresh plate of samples (combining the PCR product and size standard). 
• High salt concentration in the sample may cause broad peaks. Run a size standard-only plate to determine if it is a sample issue. If not, replacement of the polymer, buffer and/or array may be necessary. 
• Leaks on the system can divert the current and slow the migration of the sample. Check for leaks at the capillary/capillary array entering the pump or polymer block and the tubing entering the polymer block. 
• If the issue persists, please contact Technical Support at techsupport@thermofisher.com for additional troubleshooting. 

The size of the PCR product may not match the size confirmed by sequencing because a size standard is used to extrapolate the base-pair sizes of the sample product peaks. The fragment analysis software uses the size standard in each sample to create a standard curve for each sample. It then determines the relative size of each dye-labeled fragment in the sample by comparing fragments with the standard curve for that specific sample. The mobility of the fragments is affected by the sequence composition, the fluorescent label, and electrophoresis conditions. Although the PCR product size may not match that confirmed by sequencing, once the size has been established, the precision, or reproducibility, will be consistent for a given fragment.

If the sequence composition, fluorescent label, electrophoresis conditions, or size standard are changed, this will impact the relative size of the fragment. 

If the fragments have shifted in size and the same capillary electrophoresis (CE) instrument, dyes, and run module are being used, the polymer should be checked to see how long it has been on the instrument and whether it has expired. The polymer can be kept on most CE instruments for 7 days, with the exception of the 3500/3500xL Genetic Analyzers where the polymer may be on the instrument for up to 14 days if the lab temperature is below 25 degrees C. 

• As the size of a fragment is calculated based on the size standard with which it co-migrates, dye-labeled DNA fragments can yield different sizes when run with a different instrument, polymer, capillary array length, or size standard. 
• Check to see whether the polymer, buffer, or array has expired and/or is degraded. If so, they will need to be replaced.
• Check that the analysis parameters, analysis method, and size standard selected in the analysis software are the same. 
• It is also possible to observe slight shifts in sizing if the lab temperatures differ. This will also impact the migration of the sample. 
• If multiple injections of the control yield the same size, then it may be necessary to note the offset in sizes for a given fragment as this will confirm the instrument precision. 

In GeneMapper™ Software, “failed sizing” is a result of the software being unable to identify the size standard peaks as defined by the selected size Standard.Please confirm that the size standard used in the sample matches the size standard selected in GeneMapper™ Software. The Size Match Editor function in GeneMapper™ Software allows you to view the size standard peaks sizing assignments. In the Size Match Editor window, the size standard peak heights can be reviewed, the user can determine if all the peaks in the size standard are present, or if there are any extraneous peaks in the same color as the size standard that are present. The Size Match Editor can be accessed by highlighting the failed samples in the Samples tab, and then going to the Analysis pull down menu > Size Match Editor. 

• Within the GeneMapper™ Software’s Analysis Method, the Peak Detector tab contains the minimum peak height setting for each color (blue, green, yellow, red, purple, and orange). If the size standard peaks are below the minimum peak height setting on the Y-axis, typically 50, the software will ignore these peaks (this threshold is different for fragment analysis that occurs in the 3500 Data Collection software). Although this setting may be altered, it may be necessary to troubleshoot further before doing so. If the sample has strong signal and the size standard is weak, the sample may be preferentially injecting into the capillaries. The sample may have a high salt concentration and/or robust amplification. To confirm, a size standard-only sample should be run to confirm the performance of the size standard alone. If the signal intensity of the size standard improves, the sample may require desalting or less PCR product should be used. If the size standard-only sample still shows low signal, the issue may be possible degradation of the size standard or the Hi-Di™ Formamide and they may need to be replaced. It may also be a sign that the capillary array is old or clogged.
• If there are any extraneous peaks, this can be due to pull-up peaks from overloaded samples. Extraneous peaks in the same color as the size standard dye label may interfere with the sizing algorithm in the analysis software. The sample can be reduced to prevent the pull-up peak from overloaded samples. If the sample is not overloaded, please confirm that the fluorescent dye in use is part of the dye set selected. The spectral calibration may also need to be re-run. 
• If the larger peaks in the size standard are missing, it can also cause the sizing analysis to fail. This could be sample-related where smaller ions and PCR products are preferentially injected into the capillaries and out-compete the injection of the larger products. To confirm, the size standard should be run without the sample. 
• If a size standard-only injection is performed, the larger size standard peaks may be missing due to slower migration of the sample. Slower migration can be due to some of the following:
  • Degraded or expired polymer
  • Old array or capillary
  • Old or incorrect concentration of the buffer
  • Old or expired HiDi™ Formamide
  • Colder ambient temperature
  • Oven temperature too low

If the issue persists, the instrument run time can be increased in order to allow for longer collection time of the largest size standard peaks. However, if the peak resolution is also broader than expected, this may suggest an instrument issue and a service call should be opened. 

• If the smaller peaks in the size standard are missing, they may be masked by the primer peak. The easiest fix is to go to the GeneMapper™ Manager, Size Standard tab, select the size standard definition you are working with and click the “Save As” button at the bottom. Rename the new standard and edit it by deleting any size smaller than 50 bp. Click OK, then Done and change the size standard in your project and re-analyze the data. 

The Size Match Editor will allow overriding of the sample quality but it is important to confirm the size standard peaks are correctly identified. Otherwise, the peak sizes of the PCR products may be shifted. To override the sizing quality in the Size Match Editor, click on the “Override SQ” button on the top, middle of the window. 

If the sample has strong signal and the size standard is weak, the sample may be preferentially injecting into the capillaries. The sample may have a high salt concentration and/or robust amplification. To confirm, a size standard-only sample should be run to confirm the performance of the size standard alone. If the signal intensity improves, the sample may require desalting or less PCR product should be used. If the size standard-only sample still shows low signal, the issue may be with possible degradation of the size standard and it may need to be replaced. 

If a size standard-only run is performed and is missing the smaller fragments, degradation of the size standard may have occurred. Please check the expiration date either on the box or the Certificate of Analysis (COA). The size standard should be stored at 4-8 degrees C. Freezing of the size standard will cause loss of the smaller products and may also result in dye breakdown. The size standard should be replaced if expired or stored improperly. If the smaller peaks are missing, they may also be masked by the primer peak. The easiest fix is to go to the GeneMapper™ Manager, Size Standard tab, select the size standard definition you are working with and click the “Save As” button at the bottom. Rename the new standard and edit it by deleting any size smaller than 50 bp. Click OK, then Done and change the Size Standard in your project and re-analyze the data. 

If the larger peaks in the size standard are missing, it can also cause the sizing analysis to fail. This could be sample-related, where smaller ions and PCR products are preferentially injected into the capillaries and out-compete the injection of the larger products. To confirm, the size standard should be run without the sample. 

If a size standard-only injection is performed, the larger size standard peaks may be missing due to slower migration of the sample. Slower migration can be due to some of the following:

• Degraded or expired polymer
• Old array or capillary
• Old or incorrect concentration of the buffer
• Old or expired HiDi™ Formamide
• Colder ambient temperature
• Oven temperature too low

If the issue persists, the instrument run time can be increased in order to allow for longer collection time of the largest size standard peaks. However, if the peak resolution is also broader than expected, this may suggest an instrument issue and a service call should be opened. 

This is normal as the SNaPshot ™ control shown in the manual is from a 3100 Genetic Analyzer and POP-4™ polymer. Dye-labeled DNA fragments can yield different sizes when run with a different instrument, polymer type, capillary array length, or size standard. Although the control sizes may not match that in the protocol, once the size has been established, the precision, or reproducibility will be consistent for a given fragment. 

Due to the influence of the dye on the mobility shift of the DNA fragments, the reported sizes will differ by a few bases from the actual sizes. This is particularly true with the shorter fragments as the relative contribution of the dye is greater. It is also recommended that you have a tail on the primer with a 5–7 bp difference between them to further separate the samples so there is no overlap.

The extra peaks could be due to incomplete removal of PCR primers, incomplete removal of dNTPs from the PCR reaction, or incomplete removal of the fluorescently labeled ddNTPS from the SNaPshot™ reaction. Residual PCR primers and dNTPs will both participate in the SNaPshot™ reaction. For the PCR purification, please use fresh SAP and Exo1 or use another method of PCR purification. To address incomplete removal of fluorescently labeled ddNTPS, use fresh CIP or SAP.