AbstractMotivationAlignment-free, k-mer based genotyping methods are a fast alternative to alignment-based methods and are particularly well suited for genotyping larger cohorts. The sensitivity of algorithms, that work with k-mers, can be increased by using spaced seeds, however, the application of spaced seeds in k-mer based genotyping methods has not been researched yet.ResultsWe add a spaced seeds functionality to the genotyping software PanGenie and use it to calculate genotypes. This significantly improves sensitivity and F-score when genotyping SNPs, indels, and structural variants on reads with low (5×) and high (30×) coverage. Improvements are greater than what could be achieved by just increasing the length of contiguous k-mers. Effect sizes are particularly large for low coverage data. If applications implement effective algorithms for hashing of spaced k-mers, spaced k-mers have the potential to become an useful technique in k-mer based genotyping.Availability and implementationThe source code of our proposed tool MaskedPanGenie is openly available onhttps://github.com/hhaentze/MaskedPangenie.