Viewing raw chromatogram data from .ab1 files

If you are interested in looking for or evaluating mixtures of residues in .ab1 or .scf chromatogram files, it is important to be able to extract the raw data from the four traces. You can open .ab1 and .scf files directly in MacVector by using the File | Open menu item or by dragging files onto the MacVector application icon. You *may* also be able to open files in MacVector by double-clicking on them, depending on which other applications you have installed on your machine. Once open, you can click on the Raw Data tab to view the trace data in tabular form;

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The “Tot. Area” column lists the total signal for each of the four traces combined at each peak position. The values are simply the signal units output by the sequencing machine. This is followed by the individual signal for each of the four traces (i.e. the “area under the curve”), along with the percentage of the total signal represented by that trace. So in the middle of a clean sequencing run, you might expect each base-called peak to represent >90% of the signal. Conversely, a SNP allele in a direct genomic sequencing run might show as an equal 50% mix of the two traces. The “mix” columns alert you to these possible mixed residues at different percentage levels. For example if you see “GT” in the 35% mix column, that indicates that both G and T represented more than 35% of the signal at that particular peak.

Finally, the entire list is displayed as tab separated values. This means that you can select the data and copy it into Microsoft Excel and each value will be placed into a separate cell so that you can perform additional statistical analysis outside of MacVector.

For more information, check out our 101 tips blog post.

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