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Troubleshooting

Here are some of the main causes of DNA sequencing problems and how to identify and overcome them.

Failed Sequencing Reaction

Electropherogram has noisy or "messy" sequence peaks with low quality scores
No or little signal in the raw data channels except for leftover dye at the beginning of the trace.
Signal strength in the raw channel usually below 100.
The sequence does not match either the expected sequence or other sequences in GenBank

Electropherogram has two or more sequence peaks at the same location. The secondary peaks may be the same height as the primary peaks down to about 20%. Lower levels of mixed signal (below 20%) are normally base- called well by the KB
The trace signal strength is good (>200U) in the raw channel
The quality scores are low with often less than 100 Q20+ bases
The base-called sequence does not match the expected sequence or any known sequence in GenBank

The trace signal is mixed in the early regions (normally before base 400) but not in the late regions
The signal drops from very high early in the trace (usually in the first 100 bases) to much lower levels
The total quality scores counts are generally low to moderate depending on the region affected

Sequence provides less than 750 Q20+ bases in electropherogram (when a longer sequence is expected)
End of the sequence is "messy"
Signal ends abruptly before base 1000 (when a longer sequence is expected)

Good quality trace signal suddenly stops, or rapidly declines
The average quality scores after the "hard stop" region are generally low
Template has a high percentage of G and/or C nucleotides, especially in the region where the stop occurred

Very high peaks in the raw data trace that fade abruptly
In the electropherogram, base calls fade before the end of the read
An excess of short fragments are generated that are preferentially injected into the capillary

Overlapping sequence following a homopolymer region due to slippage of the enzyme