Author contributions K.C. and L.P. conceived the project, led the study, and wrote the software. K.C. analyzed experimental data. All authors contributed input on measurement and visualization of genome editing outcomes and provided input on the manuscript. Competing Interests At the time of manuscript preparation, J.M.G. was a consultant for Beam Therapeutics, and now is employed by Beam Therapeutics. J.K.J. has financial interests in Beam Therapeutics, Editas Medicine, Endcadia, EpiLogic Therapeutics, Pairwise Plants, Poseida Therapeutics and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. J.K.J. is a member of the Board of Directors of the American Society of Gene and Cell Therapy. J.M.G. and J.K.J. are co-inventors on patents and patent applications that describe gene editing technologies. D.R.L. is a consultant and cofounder of Editas Medicine, Pairwise Plants and Beam Therapeutics, companies that use genome editing. Code availability.

MotivationUnique molecular identifiers (UMIs) are added to DNA fragments before PCR amplification to discriminate between alleles arising from the same genomic locus and sequencing reads produced by PCR amplification. While computational methods have been developed to take into account UMI information in genome-wide and single-cell sequencing studies, they are not designed for modern amplicon-based sequencing experiments, especially in cases of high allelic diversity. Importantly, no guidelines are provided for the design of optimal UMI length for amplicon-based sequencing experiments.ResultsBased on the total number of DNA fragments and the distribution of allele frequencies, we present a model for the determination of the minimum UMI length required to prevent UMI collisions and reduce allelic distortion. We also introduce a user-friendly software tool called AmpUMI to assist in the design and the analysis of UMI-based amplicon sequencing studies. AmpUMI provides quality control metrics on frequency and quality of UMIs, and trims and deduplicates amplicon sequences with user specified parameters for use in downstream analysis.Availability and implementationAmpUMI is open-source and freely available at http://github.com/pinellolab/AmpUMI.