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Mitochondrial metagenomics (MMG) using Illumina sequencers for mixed-species samples provides a promising tool for evolutionary and ecological studies using mitogenomes. However, the traditional assembly procedure is still computationally intensive and expensive. Here, a novel MMG pipeline was applied to different DNA extractions, one per species, and their sequence as a mixed sample for rapid mitogenome assembly is presented. Our method integrated a faster and more accurate read mapper for filtering non-mitochondrial reads. A seed-and-extend assembler for species-specific mitogenomes that detects ‘noisy species/sequences’ was also assessed. The MMG pipeline for each dataset was completed in a few hours on desktop PCs, maintaining high accuracy and completeness (COI divergence &gt;10%), except for some very closely related taxa. Particularly for closely related species, the exclusion of ‘noisy reads’ (including chimera of non-targeted species) improved the target assembly. In addition, we observed that short barcodes used as references had almost identical detection power compared with mitogenomes but required greater sequencing depth. We tested our MMG pipeline on two real and one simulated dataset to validate its high efficiency in mixed-species sample mitogenome assembly.

Advancing Mitochondrial Metagenomics: A New Assembly Strategy and Validating the Power of Seed-Based Approach