IndelQuant: A Workflow for Cost-Efficient Amplicon Sequencing Analysis of Genome-Edited Chickens

Accurate quantification of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-induced insertions and deletions (indels) is essential in chicken genome-editing studies, where low editing efficiencies and mosaicism can complicate the assessment of target-site modifications. Existing assays often involve a trade-off between affordability and the sensitivity orsequence information needed for detailed analysis. Here, we introduce IndelQuant, a cost-efficient workflow integrating sample-specific barcode PCR, pooled amplicon library construction, AdapterRemoval3-based read processing, and CRISPResso2-based indel quantification. The workflow provides graphical and tabularsummaries of editing efficiency, indel positions, indel-size distributions, and sample-level mutation profiles. We evaluated the workflow in TYR-edited DF-1 cells and genome-edited chickens. In edited chickens, IndelQuant detected tissue-specific indel signals, most below 5%, while negative controls showed background frequencies from 0 to 0.03%. These findings indicate that IndelQuant retains the sequence-level information provided by amplicon sequencing while simplifying sample processing and analysis, offering a practical approach to routine screening for low-frequency and mosaic indels in poultry genome-editing studies.