Short DNA Trace Read Lengths
Identification of short DNA sequencing reads
- Sequence provide less than 650 Q20+ bases for traces run on 36cm arrays and less than 750 Q20+ bases for traces collected on 50cm arrays under standard run conditions.
- End of the trace is "messy" with "rough" looking peaks
- Trace signal ends abruptly before base 800 on 36cm array traces and before base 1000 on 50cm array traces.
Figure 1. Example of a short/poor trace. (A) KB processed data channel data at approximately base 550. (B) Raw channel data at the same base location.
Causes of short DNA sequencing read lengths
- Too much template DNA
- Excessive dilution of the BigDye reagent
- Too little DNA
- Too much primer
- "Dirty" template DNA has been used.
- Unsequencable region reached (eg. homopolymer G)
Solutions for short trace reads
- Check the concentration of the template DNA by gel electrophoresis. Do not rely on spectrophotometer readings alone as spec reading are often inaccurate, particular with plasmid templates.
- Reduce the reaction scale rather than using a higher bigdye dilution. It is better to reduce the reaction volume rather than use very high dilution factors of the BigDye chemistry. For example, a 1:16 dilution of the BigDye (0.5 µl total ) can be achieve with modest dilution (1:4) by performing the reaction in a total volume of 5µl.
- Check to see if an unsequencable region has been reached. If an unsequencable or “hard stop” region is the cause then it may be possible to sequence though it by PCR amplifying the region using 7-deaza-deoxy guanosine triphosphate (7-deaza-dGTP), then sequencing the PCR product directly. The 7-deaza-dGTP analog disrupts the hoogsteen base pairing between successive guanosine bases and allow the sequencing DNA polymerase through the high G+C region.
- Check that the oligonucleotide primer concentration is correct. Do not rely on old primer stocks, especially those made up by your lab colleagues!
- Check that the template is clean. Consider sequencing a PCR amplified insert or switching to using a commercial plasmid miniprep kit if you are finding that many of your sequencing reactions are failing.
Return to the main DNA sequencing troubleshooting page.