5.5. Why are there ambiguity codes and N's in the paired reads of my mapping?
The presence of ambiguity codes and N's in mapped reads can both relate to the use of overlapping paired reads for which there is a conflicting base, insertion or deletion between the two reads. When paired reads overlap they will appear as one concatenated blue read, for which conflicts will be shown with an ambiguity code and insertion/deletions with an N.
Ambiguity code in mapped reads:
An ambiguity code will be assigned to any base where the two members of a pair of reads overlap, but do not agree on the base call.
N's in mapped reads:
An N will be assigned to any position where the two members of a pair of reads overlap, but there is an insert or deletion in one of the reads on the base call.
To view the conflicts in the individual forward and reverse reads you can go to the settings panel and check the box for Disconnect paired reads. When selecting the Disconnect paired reads option in the settings panel the individual forward and reverse reads will be shown revealing the deletion, conflict, and insertion.
How is this information handled by the Workbench variant callers:
The Fixed Ploidy and Low Frequency Variant Detection tools will ignore overlapping reads that do not agree about the variant base, whereas the Basic Variant Detection tool will consider the position if only one of the reads passes the quality filter.
More general information about how overlapping paired reads are handled can be found on the following manual page: