Today I ran a high
sensitivity DNA bioanalyzer chip to quality check some amplicon
libraries I was setting up for the MiSeq. In so doing, I noticed that
my peaks were coming out at the wrong sizes, for a simple reason; the
2100 Expert software hadn't correctly detected one of the ladder
peaks (the second, 50bp band).
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Note second peak from left has a peak concentration of zero! |
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It hasn't been recorded as the 50bp ladder band, instead mistakenly recorded as 39bp |
I had a look through
the user
guide and the troubleshooting
guide (see page 41) to try to re-assign the ladder peaks as you
can set the upper and lower marker using manual integration, to no
avail. A quick google and check of SeqAnswers didn't help either.
In case anyone's
struggling with a similar problem/lack of immediately searchable
answer, here's how I solved it.
The problem was really
that the software had called the two biggest peaks at a location that
really was just the biggest; presumably it looks for the upper and
lower markers first, then assigns the remaining peaks from largest to
smallest.
This is easily solved -
I just right clicked the peak (on the Peak Table tab when looking at
the electropherogram) and exclude it. After that the program
re-calculates the proper sizes, assigning the 50bp peak correctly and
outputting the correct sizes for my samples. Job done!
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The slight shoulder off the upper marker mistakenly got detected as the next peak down |
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Much better! |
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Now the band is correctly labelled |
This is probably an
obvious fix, but if (like me) you were looking for the wrong search
terms (re-assigning peaks when really I needed to exclude) hopefully
this might help you shave a little bit of time off for you!
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