WAS IT A MATch I SAW? Approximate palindromes lead to overstated false match rates in benchmarks using reversed sequences.

Overestimates of false match risk can motivate unnecessarily high score thresholds, leading to potentially reduced true match sensitivity. Also, when tool sensitivity is only reported up to the score of the first matched decoy sequence, a large decoy set consisting of reversed sequences can obscure sensitivity differences between tools. As a result of these observations, we advise that reversed biological sequences be used as decoys only when care is taken to remove positive matches in the original (un-reversed) sequences, or when overstatement of false labeling is not a concern. Though the primary focus of the analysis is on sequence annotation, we also demonstrate that the prevalence of internal palindromes may lead to an overstatement of the rate of false labels in protein identification with mass spectrometry.