![]() ![]() 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.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |