Many bioinformatics methods seek to reduce reference bias, but no methods exist to comprehensively measure it. Biastools analyzes and categorizes instances of reference bias. It works in various scenarios: when the donor’s variants are known and reads are simulated; when donor variants are known and reads are real; and when variants are unknown and reads are real. Using biastools, we observe that more inclusive graph genomes result in fewer biased sites. We find that end-to-end alignment reduces bias at indels relative to local aligners. Finally, we use biastools to characterize how T2T references improve large-scale bias.
