STEP III - Loss of Resolution Later in the GelĮven normal chromatograms stop providing accurate data after some distance along sequence:Īs the gel progresses, it loses resolution. A comparison of text sequences would probably notify you of the presence of a SNP at this particular location. **Note that the peak was called an 'N' by the basecaller. Both peaks are present, but at roughly half the height they would show if they were homozygous. In this case, one allele carries a C, while the other has a T. Here is a great example of a PCR amplicon from genomic DNA, with a clear heterozygous single-nucleotide polymorphism (SNP). If you want to be sure you've detected all of the polymorphic positions, you should be using a computer program to scan your chromatograms! Realize, too, that it's easy for a human to miss these. **Note that the basecaller may list that base position as an 'N', or it may simply call the larger of the two peaks. This is common when sequencing a PCR product derived from diploid genomic DNA, where polymorphic positions will show both nucleotides simultaneously. No harm done, in this case the sequence is fine.Ī single peak position within a trace may have but two peaks of different colors instead of just one. **Note the extra space between the letters G and A (nt's 271 and 272) corresponding to the mis-spaced peaks just below them. Often, it is ignored by the basecaller, as in this example at right: A common one is a G-A dinucleotide, which leaves a little extra space between them. Some sequencers have predictable errors in base spacing. Nucleotides that have been erroneously inserted into a sequence will often appear to be oddly spaced relative to their neighboring bases, often too close. At the same time, watch for mis-spaced letters in the text sequence along the top. One good way to detect artifacts or errors in a sequencing chromatogram is to scan through it, looking for mis-spaced peaks. Quickly scan the gel for extremely small peaks, 'N' calls, and any mis-spaced peaks or nucleotides. Such mis-calls can occur even in the most error-free regions of the gel. Occasionally, the computer will call an 'N' when a human would be confident in making a more specific basecall. Most often, this occurs when the basecaller calls a specific nucleotide, when the peak really was ambiguous and should have been called as 'N'. Sometimes the computer will mis-call a nucleotide when a human would have identified a different nucleotide.
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