Geneious has a Compare Annotations function which allows you to filter SNPs based on their overlap with another annotation track or annotation type. We will use this function to filter out SNPs that are in regions of low coverage*.
In Exercise 3 we created an annotation track called "Coverage" to identify regions where coverage falls to below two standard deviations from the mean coverage. Compare annotations will allow us to exclude annotations from the Variants track that fall within the low coverage annotations.
Select your reference sequence. This should now have the tracks you created in the earlier exercises displayed on it. Go to Annotate & Predict → Compare Annotations. You can specify the annotations you wish to compare in the Annotation Types panel. For Set A select "Polymorphism" "on track Variants: yghJ paired Illumina reads", and for Set B select "Low" "on track Coverage: yghJ paired Illumina reads". Under Comparison, uncheck Names must match, as the polymorphism and coverage annotations have different names. Check Allow intervals to partially match..., as this will return polymorphisms such as indels that are only partly within a low coverage annotation.
Under results check A-B. This will return polymorphism annotations that do not overlap with coverage annotations - the venn diagram to the right, and the "Example" panel displays this graphically. The window should now look as in the figure below.
Click OK, and you should now see a third annotation track on your reference sequence called "Variants: yghJ paired Illumina reads - Coverage: yghJ paired Illumina reads". This track has fewer polymorphism annotations than the original Variants track. Scroll along to regions of the sequence marked as low coverage and you weill see that there are a number of polymorphism annotations in the original Variants have been excluded from the new track.
Click Save to record the new track on your reference sequence.
*Note: For demonstration purposes in the tutorial, we've filtered out low coverage SNPs using Compare Annotations. However, you can set up the SNP finder to automatically filter out low coverage SNPs without needing to perform this step. To do this, check the Minimum Coverage box in Find Variations/SNPs and enter the minimum coverage required for a SNP to be called.
Congratulations, you have now completed the Mapping and SNP calling tutorial.