Quantification of spliced and unspliced transcripts by velocyto is inaccurate for 5'-sequencing data
RNA velocity allows for the prediction of future states of individual cells based on their current transcriptional activity. The technique depends on the separate quantification of spliced and unspliced transcripts in single-cell RNA-sequencing data, first introduced by velocyto in 2018. Since its introduction, significant advances have been made in the field, including new protocols by 10x Genomics that enable sequencing from the 5-end of mRNA molecules instead of the 3-end. Despite these advances, velocyto has not been updated since its release but is still commonly used with these new protocols. In this study, we demonstrate that velocyto cannot accurately detect the reversed direction of transcripts, leading to incorrect count assignments. By comparing velocyto to alevin-fry, a quantification method compatible with 5-sequencing data, we show that this limitation can result in substantial deviations in inferred velocities and differing interpretations. Therefore, we do not recommend the use of velocyto with 5-sequencing data.