I performed an alignment of reads from Drosophila simulans (a sister species to melanogaster) against a modified Drosophila melanogaster reference genome where sites that mismatched with simulans were set to N (I had a specific reason for using this alignment).
Afterwards, the same modified reference genome was used for variant calling using GATK's unified genotyper. It didn't occur to me until after the fact that this might lead GATK not to return genotypes at the sites that I set to N. Does UnifiedGenotyper skip/ignore sites where the reference genotype is N, even if there is strong support for particular nucleotides at that site, or can it still identify genotypes at these sites in the final vcf (i.e. can it treat NN as reference and e.g. AA or AG as "variant")?
Does UnifiedGenotyper skip/ignore sites where the reference genotype is N
yes. The variant is in YOUR data, not in the reference where 'N' is for unknown (centromere etc...)
Just to clarify, I have set additional sites in the reference genome to 'N', which means (based on your statement) that these will not be genotyped by GATK regardless of what and how many bases are found in the reads at this site?
As a follow-up, is it possible to get a variant call with UnifiedGenotyper if rather than N, I have one of the ambiguity codes (e.g. R, Y, B etc) in the reference genome?
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Whether N -> A/T/G/C represents a variant is heavily dependent on the tool itself, and I know most tools don't. Since you don't see it in your vcf, that means that unified genotyper doesn't like it.
A bit digression. This unifiedgenotyper is so old that ppl have switched to haplotypecaller for 10+ years.