Sanger Sequencing Troubleshooting

This section highlights the problems we most commonly see with DNA sequencing, the possible causes and the recommendations, based on our experience and the information from the following sources:

Nucleics.com – DNA Sequencing Troubleshooting

Australian Genome Research Facility – Troubleshooting Guide – Sanger Sequencing

The University of Edinburgh – The Gene Pool – Sanger Sequencing Troubleshooting Guide

Oregon State University – Centre for Genome Research & Biocomputing – Troubleshooting

The University of Hong Kong – Centre for Genomic Sciences – Troubleshooting

 

The troubleshooting guide includes:

 

Pictures of the raw data trace and the analyzed electropherogram, as displayed by the Sequence Scanner software, are provided along with the possible causes and the recommendations.

We strongly encourage all users to examine the raw data trace for signal intensity (relative fluorescence units, RFUs, displayed on the Y axis) and the electropherogram trace for sequence quality, when analyzing and troubleshooting the results. The software re-scales the raw data to improve peak visibility in the electropherogram view. Therefore, peak height in the electropherogram trace should not be used as an indicator of signal intensity.

 

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