Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide
23
Note that the characteristic incorporation effects of thermally stable DNA Polymerase
and the WellRED™ dyes can be helpful in diagnosing prepeaks from true peaks. For
example; a T followed by a T always has a much larger signal for the first T than the
second T (Figure 17). A small T followed by a large T therefore, indicates that the
small T is a prepeak and not the correct sequence.
Corrective Actions:
The only way to rectify this problem is to obtain high quality oligonucleotides for use
as sequencing primers.
1.
If you obtain sequencing primers from an external source (oligo vendor, core
facility, etc.), request that your oligonucleotide supplier use the highest quality
DNA synthesis conditions. Also note that oligonucleotide purification methods
that rely on chromatography can not completely separate the n-1 from full-length
oligonucleotides and therefore can not “rescue” a contaminated primer
preparation.
2.
If you synthesize sequencing primers using an automated DNA synthesizer, use
the “High quality” settings on your instrument. This usually involves using
increased amount of “capping” reagents and chemical coupling times. As is
noted above, oligonucleotide purification methods that rely on chromatography
can not completely separate the n-1 from full-length oligonucleotides and
therefore can not “rescue” a contaminated primer preparation.
3.
If you have a software version of 3.0 or higher, check the “pre-peak removal”
check box in “Analysis Parameters” upon reanalysis of data. This will help to
alleviate base call insertions due to n-1 primers. Although, it may not correct for
all possible insertions if the sequencing primer is too greatly contaminated with
n-1 products.
Figure 17: No n-1 primer insertions
No n-1 insertions present
