Identifying sequencing runs with excess collection times
- The trace has very large peak (void peak) at the end of the trace which is easily 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 the raw channel data of a trace using an excessive run time. Note the void peak at the end of the trace. This is only visible in the raw data.
Causes of sequencing runs with excess collection times
- Using an ABI instrument data collection time that exceeds the resolution of the sequencing matrix under a given run condition. This causes all the sequencing fragments larger than a certain size to elute simultaneously in a single large or void peak (Figure 1).
- Using high run voltage. The higher the run voltage the shorter 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. The exceptions are if you are using PeakTrace and the trace is high quality to the end of the traces.
- 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 of your traces in the time available.
For more information on automated QC tracking of sequencing traces please visit the QualTrace III DNA sequencing analysis software page.
Return to the main DNA sequencing troubleshooting page.