Hi all,
I am attempting to carry out microsatellite genotyping, using Gene Mapper 5 to analyse data output by the ABI 3500XL Genetic Analyser, but I am encountering an error message I have never seen before despite using this method for several months previously. Upon importing sample data into Gene Mapper, and selecting 'Analysis Method', 'Panel' and 'Size Standard' (all of which have been used without issue in the past), I find that the 'Display Plots' and 'Size Match Editor' options are unavailable for some samples. If I attempt to select multiple samples, including these strange samples, I receive the following error message:
"Some selected sample(s) do not contain analysis data. Those sample(s) will not be shown."
The '.fsa' files for these samples are smaller than for other samples (185 KB vs 265 KB), however, these samples can still be analysed, along with the other samples. Following analysis, size standard peaks are suddenly available for these problem samples. These ladders have more peaks labelled than the selected size standard should allow them to (they should not have the '250' peak labelled but do).
Is anyone able to offer some advice as to what is happening here and what may be causing these strange occurrences, please? My first assumption was that the formamide or size standard being used had degraded or insufficient size standard was being included per sample but, firstly, individual tubes of each has been found to work for one run but not for another on a different day and, secondly, an absence of size standard should be met with a 'No sizing data' message.
Any advice you can offer would be greatly appreciated.
Many thanks,
Ian Wilson
Hi Muhammad Ahmad
I include here a few photos to try and illustrate the problem. In screenshot 1, you can see the error message received when I try to view the size editor for problematic samples. Screenshot 2 shows that GeneMapper does not display size standards for these samples. I then pressed the 'Analyze' option and screenshot 3 shows that the samples missing size standards in screenshot 2 now have size standards. Interestingly, you may note that the size standard I instructed GeneMapper to use omits the '250' peak (screenshot 2), however the ladder in screenshot 3 has this peak. As such, I assume that GeneMapper cannot be taking its peak labels from the size standard it was instructed to.
Any comments or advice would be gratefully received, thank you.
Ian
there are some peaks in your size standard which are not labeled and gen mapper is confusing it. If you think your size standard is fine you can over ride the quality by pressing over ride quality button and the see if it works.
Kind Regards
Ahmad
Muhammad Ahmad, those peaks are intentionally missed out to try and avoid mis-labelling of peaks. Confusion over quality of peaks wouldn't cause the "Some selected sample(s) do not contain analysis data" error message and an absence of a viewable size standard though, would it? You'd surely just get a low quality score or a 'No size standard' image?
but you need to press override quality otherwise it will not take it as standard
i was wondering also this is also a pipetting mistake that somehow you forgot to add your sample into it. Perhaps you might rerun these four samples. Sorry If iam wrong/
No, that's okay, I've wondered the same thing myself. But, if it's a pipetting error, it's more likely to be a lack of size standard than samples based on the numbers across the whole plate.
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