DNA Sequencing Troubleshooting

Guide for troubleshooting automated DNA sequencing problems on the ABI 3730, ABI 3130 and ABI 3100 along with helpful tips for improving the sequencing of DNA and RNA

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Troubleshooting DNA Sequencing Problems

Automated DNA sequencing is one of the most common and robust techniques performed in molecular biological laboratories. Unfortunately, it does not always work and when it doesn't it can be very difficult to work out what went wrong. Fortunately, most failed (or sub-optimal) DNA sequencing results have only a fairly limited number of causes. To help in the troubleshooting of sequencing problems we have created a series of guides for identifying the most common causes of various sequencing problems. These guides also includes tips on how to overcome each problem type, along with more general tips for improving DNA sequencing quality.

If you have any comments or questions about these guides then please contact our as we would love to hear your suggestions.

How to Identify the Causes of DNA Sequencing Failures

Identifying the cause of a poor DNA sequencing result can often be very difficult as a particular sequencing problem may have many different underlying causes, or be the result of multiple interacting factors. Often the only way to work out the real cause of a particular problem is to perform a process of elimination.

This process can be greatly simplified by visually examining both the raw and processed data chromatograms of the problematic sequencing traces. In the following guide we have provided detailed information on the most commonly observed sequencing problems, along with suggestions on their most likely causes. The causes are listed in order from the most common to least common. We have also included solutions (where known) on how to overcome each sequencing problem type.

An alternative to manual inspection (which becomes very labor intensive if you are running more than a few traces) is to use an automated trace analysis system like our QualTrace DNA sequencing QC software. QualTrace will automatically scan the traces for nine different sequencing problems, and because it work by analyzing the raw data, is able to be used with any base caller.

To let people see how QualTrace can help in DNA sequencing troubleshooting we have created a free, online version of QualTrace where you can upload your own traces and have QualTrace analyze them for any problems.

The Most Common Automated DNA Sequencing Problems

The following guides give examples of the major causes of sequencing problems and describes how to identify them and solve the underlying problems.

Failure of the DNA sequencing reaction

Mixed signal (multiple peaks) in the trace

Short read lengths and poor quality data

Poorly resolved trace peaks "blurry peaks"

Excessive free dye peaks "dye blobs" in the trace

Misshaped or "noisy" in the trace peaks

Primer dimer formation in the sequencing reaction

Sharp signal spikes in the trace chromatogram

DNA Polymerase slippage on template mononucleotide regions

Sequence stops in "difficult" template regions

Breakdown of the DNA sequencing BigDye chemistry

Insertion or deletions (indels) in the DNA template

Chimeras and sequencing rearrangements

Delayed start of trace signal in the raw signal channel

A "G dye blob" at approximately base 190 and 400

Excessive trace data collection times