Abstract 2425 Single-Cell mtDNA Variant Analysis with scMOCHA

Mitochondria, double-membrane organelles housing their own DNA genome (mtDNA), play a pivotal role in generating over 90% of cellular energy through oxidative phosphorylation (OXPHOS). mtDNA variants are closely linked to various human diseases. Exploring deeply about mtDNA variants can offer valuable insights into their association with human physiology and pathology. Single-cell RNA sequencing (scRNA-seq) or Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) data typically encompass 5-25% of reads mapping to the mtDNA genome. Unfortunately, these mtDNA reads are often excluded during downstream analyses to enhance clustering and cell type identification accuracy. The current lack of robust tools for mtDNA data analysis in single-cell genomics prompts the need for a comprehensive workflow. Thus, our objective is to build an optimized mtDNA variant analysis workflow for scRNA-seq and scATAC-seq data, leveraging diverse datasets. In this study, we download scRNA-seq and scATAC-seq datasets from donors with diverse backgrounds for tool development. During analysis, we introduce a step to include cells with higher mtDNA reads without compromising clustering accuracy after an initial analysis (read QC, alignment to both human nuclear and mitochondrial genomes, normalization, etc.). Then, we test the impact of different mtDNA% thresholds on clustering to enhance mtDNA reads and genome coverage, facilitating accurate mtDNA analysis and variant calling. After that, we modified a recently published mtDNA analysis script for scRNA-seq data, encompassing mtDNA mapping, variant calling, and mtDNA gene expression analysis, and incorporated it into standard nuclear gene expression analysis. Finally, we optimize the workflow by testing it on scRNA-seq datasets for robust mtDNA analysis. Our newly developed tool, scMOCHA, addresses a critical gap in mtDNA analysis in single-cell genomics. Overall, this workflow aims to empower researchers to gain novel insights into mtDNA variants and their association with human pathology. Organizations: Children's Hospital of Philadelphia (CHOP), St. Mary's University, Grants: NIH U01 HL166058, NIH HuBMAP Underrepresented Student Internship Extension Program