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Cryo-mtscATAC-seq for single-cell mitochondrial DNA genotyping and clonal tracing in archived human tissues
Maren Salla1,2,3, Benedikt Obermayer4, Marie Cotta1,2,5
1Berlin Institute of Health at Charité Universitätsmedizin Berlin, Berlin, Germany.
Biorxiv : the Preprint Server for Biology
|September 26, 2025
Summary
A new method, Cryo-mtscATAC-seq, allows high-throughput clonal tracing in frozen human tissues. This technique analyzes mitochondrial DNA (mtDNA) mutations at single-cell resolution, expanding clonal analysis to archival samples.
Area of Science:
- Genomics
- Cell Biology
- Biotechnology
Background:
- High-throughput clonal tracing in human samples uses somatic mitochondrial DNA (mtDNA) mutations.
- Fresh-frozen specimens are valuable but difficult to use for single-cell multiomics.
- Existing methods limit clonal analysis to fresh tissues.
Purpose of the Study:
- To develop a method for high-throughput clonal tracing in frozen human samples.
- To enable single-cell resolution mitochondrial genotyping in archival specimens.
- To apply this method across diverse human tissues and diseases.
Main Methods:
- Cryo-mtscATAC-seq: Isolates nuclei with mitochondria from frozen samples.
- Single-cell ATAC-seq: Analyzes chromatin accessibility and mtDNA mutations.
- Mitobender: Computational tool to reduce ambient mtDNA contamination.
Main Results:
- Applied Cryo-mtscATAC-seq to brain, glioblastoma, neuroblastoma, and aorta.
- Revealed clonal gliogenesis and microglial expansions in ALS.
- Identified persistent OPC-like clones in GBM recurrence and smooth muscle cell proliferation in aorta.
- Detected mtDNA depth heterogeneity post-neuroblastoma chemotherapy.
Conclusions:
- Cryo-mtscATAC-seq enables mtDNA genotyping in archival frozen specimens.
- The method is broadly applicable across various human tissue types.
- Expands research into cell state-informed clonality in health and disease.
