DNA sequencing traces with indels
Identification of template insertion and deletions problems
- The trace trace is excellent before the indel region and mixed after the indel region (Figure 1).
- The trace often as leading or trailing 'shifted' peaks where a small peak is found before or after a large peak (Figure 2).
Figure 1. Example of indel trace. Note the mixed sequencing after the seven T run.
Figure 2. Example of 'shifted' peaks due to a template indel. Note the small G peak preceding the large single G peak present after the nine T run.
Causes of template insertion and deletion problems
- Direct sequencing of PCR products derived from polymorphic templates. The most common cause of this is when directly sequencing PCR products amplified from diploid templates with heterozygous regions.
- Random mutation has occurred during the cloning process or during plasmid mini prep process (usually during the colony picking stage). This is much more likely to occur if the cloned template is toxic or unstable in the E. coli host.
Solving template insertion and deletion problems
- Clone the PCR products before sequencing if the template is heterozygous.
- If the template is unstable in E. coli try cloning into a low copy vector or using a different strain.
- Sequence from the other direction using a reverse primers. This option is more viable for small inserts.
Return to the main DNA sequencing troubleshooting page.