A simple centrifugation protocol for metagenomic studies increases mitochondrial DNA yield by two orders of magnitude

DNA (meta)barcoding is applied to study biodiversity and is available for standardized biodiversity assessments. However, it suffers from PCR bias, which can lead to the loss of specific taxa. PCR ‐free techniques such as shotgun metagenomics are therefore thought to be more suited for biodiversity assessments, but are currently limited by incomplete reference libraries. The technique of “mitogenome‐skimming” or “mitogenomics,” in which complete mitochondrial genomes are sequenced, is ideal to bridge the techniques of (meta)barcoding and metagenomics. However, without the enrichment of mitochondria, roughly 99% of all sequencing reads are of non‐mitochondrial origin and mostly useless for common applications, e.g. species identification. Here, we present a simple centrifugation protocol that leads to an average 140‐fold enrichment of mitochondrial DNA . By sequencing six ‘mock’‐communities—comprising the freshwater taxa Corbicula fluminea , Gammarus roeselii and Hydropsyche exocellata each—we recovered whole mitochondrial genomes of these species and the acanthocephalan endoparasite Pomphorhynchus laevis . The enrichment protocol will speed up building reference libraries for whole mitochondrial genomes, as dozens of species can be sequenced on a single MiSeq run. Subsequently, it will also allow biodiversity assessments, using mitogenomics at greatly reduced costs in comparison to mitogenomic approaches without prior enrichment for mitochondria.