"Top-Heavy" Data
Identification
- 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
Causes
- Too much template used in the sequencing reaction
- Too much primer used in the sequencing reaction
Overcoming Problem
- Check template concentration
- Check primer concentration (use 3.2 pmol in sequencing reaction)