Thursday, 8 August 2013

Re-assigning incorrect ladder peaks on the bioanalyzer

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). 
Note second peak from left has a peak concentration of zero!

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!
The slight shoulder off the upper marker mistakenly got detected as the next peak down

Much better!

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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