Abstract
Organelle genomes govern bioenergetics and stress responses, yet bulk sequencing masks heterogeneity. We establish single-organelle DNA-sequencing by adapting a droplet-based workflow to mitochondria and chloroplasts, using a sorbitol-based isolation that preserves organelles for encapsulation, amplification, and sorting. In Arabidopsis thaliana leaves, 313 of 384 single-organelle amplified genome libraries passed quality control (261 mitochondrial, 52 chloroplast). Genome coverage breadth at 1000 × sequencing depth showed medians of 59.5% and 84.5%, with maxima of 97.2% and 100%. Read-depth profiles were more fragmented for mitochondria and more uniform for chloroplasts, consistent with mitochondrial heterogeneity. This workflow enables analyses of recombination, rearrangements, and heteroplasmy.