Single-Cell Indel Detection Enhances Genetic Ancestry and Cellular Lineage Analysis

Small insertions and deletions (Indels) provide critical information for cancer genomics and clonal evolution, yet their detection from single-cell sequencing (SCS) such as scRNA-seq and scATAC-seq remains challenging due to sparse coverage, alignment artifacts, and RNA editing. Here, we present Monopogen-Indel, a bioinformatics framework for accurate germline and somatic Indel detection through haplotype-aware variant calling, dynamic template matching in repetitive regions, and cell-population-based allele segregation analysis. We validated germline Indel detection in human retina snRNA-seq with matched bulk whole genome sequencing (WGS). Monopogen-Indel detected 41,000-45,000 germline Indels per sample, with >70% precision and >90% genotyping accuracy. Using 65 heart left ventricle snATAC-seq samples, indel-based global ancestry inference segregated genetic ancestry comparably to SNVs, establishing indels as an independent marker of genetic diversity in SCS. In scRNA-seq from 43,717 cells across four anatomic sites of a patient with high grade serous ovarian cancer (HGSOC), Monopogen-Indel identified ~50,000 germline Indels per sample at 86% WGS-validated precision and an average of 1,040 de novo Indels per sample. Approximately 50% of de novo Indels reflected biological sources, including variants WGS variants, RNA editing, and cell-type-specific patterns. Somatic SNVs were correctly restricted to CD45 ^- cells, indicating high specificity in delineating somatic from germline variants. In summary, Monopogen-indel is the first framework dedicated to indel detection from SCS and expands the utility of existing single-cell data for population genetics and cancer evolution. The module is integrated into Monopogen repository https://github.com/KChen-lab/Monopogen .