Negative Results in McDonald–Kreitman MK test results

Question

[18d10fee]

Hi everyone,

I’m running McDonald–Kreitman (MK) tests across several thousand genes to estimate alpha (the proportion of adaptive substitutions).

After filtering out genes with zero values for Dn, Ds, Pn, or Ps, I still observe the following pattern:

• ~80% of genes are not significant (p > 0.05)
• Among the significant ones, ~60% have positive alpha and ~40% negative alpha
• Some alpha values are highly negative (e.g. –24)
• Alignments are codon-based and appear fine upon inspection
• Polymorphism frequency threshold = 0.1

I expected a stronger signal of positive selection overall (especially in sex-biased genes), but instead I see a predominance of non-significant and negative results.

My questions:

1. Is this distribution of alpha (many insignificant, some strongly negative) normal for large-scale MK datasets?
2. Could alignment quality or population grouping errors produce such negative alpha values?
3. Are there known biases (e.g., low polymorphism, slightly deleterious variants, demography) that could explain this pattern?

Any insights or experiences with large MK test datasets or codon-based alignments would be greatly appreciated.

Thanks!