Breakdown of the DNA sequencing chemistry
Identification of sequencing chemistry breakdown
- The traces show a progressive decline in average quality the long the tray sat on the instrument (ie sample run straight after the sequencer is loaded show better overall quality than samples run after sitting in the machine stack for 12+ hours.
- Reinjection of the sample after sitting in the instrument show poorer quality the original injection traces.
- Signal strength drops off rapidly in the raw channel ("ski-slope traces").
- Automatcally track the average quality across the tray by using software like Qualtrace. This will allow you to identify chemistry breakdown problems early.
Causes of sequencing chemistry breakdown
- Storage of the cleaned up DNA sequencing sample in solution of low pH for extended periods. This is occurs particularly when samples are resusupended in water. The pH of pure water drops to below pH 5.5 when exposed to air due to absorbence of CO2. This is not generally a problem is the sample are loaded straight away, however, if they sit on the machine overnight before the samples are loaded then they are quite likely to be damaged.
- Nuclease contamination. This is more likely to be a problem with samples resuspended in water rather than EDTA.
Solving sequencing chemistry breakdown
- Resuspend the DNA sequencing samples in 50 µM EDTA, pH 7.4. It is important that the 50 µM EDTA solution's pH is adjusted AFTER dilution from stock EDTA solution.
- Only resuspend sample just before injection.
- Only load one or two trays onto the machine at a time.
Overall, the simplest way to solve this problem is to use 50 µM EDTA ph7.4 to resuspend the samples. This approach also has the advantaged of inhibiting nuclease activity as well as preventing overloading of the sequencer capillaries.
Return to the main DNA sequencing troubleshooting page.