Trace collection time was too long
Identifying sequencing runs with excess collection times
- The trace has very large peak at the end of the trace which is visible in the raw channel (Figure 1).
- The trace peaks are very small in the processed channel.
- The quality scores are generally low with many obvious sequencing mistakes.
Figure 1. Example of a trace collected for an excessive time. (A) Processed data channel. (B) Raw channel data. Note the large signal peak at the end of the trace raw channel chromatogram.
Causes of sequencing runs with excess collection times
- Using an ABI instrument data collection time that exceeds the resolution of the sequencing matrix under the particular run condition. This causes all the sequencing fragment large than a certain size to elute simultaneously in a single large or void peak (Fig 1B).
- Using too high a run voltage. The higher the run voltage the short the "simultaneous elution" fragment size or void peak will be.
Solving runs with excess collection times
- Reduce the collection times. Unless there is a very good reason it is best to not increase standard run times by more than about 3 to 5 minutes.
- Reduce the run voltage. This will also increase the resolution of the latter part of the trace and improve read length. Aim to use the lowest run voltage that will allow you to process all you traces.
For more information on automated QC tracking of sequencing traces please visit the QualTrace DNA sequencing analysis software page.
Return to the main DNA sequencing troubleshooting page.