TY - JOUR T1 - A simple centrifugation protocol leads to a 55-fold mitochondrial DNA enrichment and paves the way for future mitogenomic research JF - bioRxiv DO - 10.1101/106583 SP - 106583 AU - Jan Niklas Macher AU - Vera Zizka AU - Alexander Martin Weigand AU - Florian Leese Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/02/08/106583.abstract N2 - 1. DNA-based methods are increasingly used to study and monitor biodiversity and DNA (meta)barcoding is readily available for standardised assessments. However, (meta)barcoding suffers from bias introduced by PCR and the use of primers, which can lead to the loss of taxonomic groups. PCR-free techniques such as metagenomics are therefore thought to be more suited for unbiased biodiversity assessments but currently suffer from the problem that reference libraries are poorly equipped with genomic information.2. The technique of ‘mitogenome-skimming’ or ‘mitogenomics’, for which mitochondrial genomes are sequenced, is ideal for bridging the techniques of (meta)barcoding and metagenomics, as reference libraries for mitochondrial genes are relatively well established. However, without the enrichment of mitochondria prior to DNA extraction and sequencing, roughly 99 % of reads are of non-mitochondrial origin and therefore mostly useless for species identification.3. Here, we present an easy to use centrifugation protocol that leads to an 55-fold enrichment of mitochondrial DNA. We sequenced six mock communities, each containing the freshwater invertebrate species Corbicula fluminea, Gammarus roeselii and Hydropsyche exocellata, and could recover the whole mitochondrial genomes of these species with a minimum coverage of 2417-fold. In addition, we were able to sequence the whole mitochondrial genome of the acanthocephalan endoparasite Pomphorhynchus laevis, which further strengthens the mitogenomics approach as unexpected taxa, for which primers have not been designed, are often not found by (meta)barcoding approaches.5. Correlation between body mass and sequence number was moderate. Thereby analyses highlight potential problems with species that have a high weight due to their shells, but produce less than expected mitochondrial reads. The here presented protocol will greatly speed up the building of reference libraries for whole mitochondrial genomes, as dozens of species could 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 mitochondrial enrichment. ER -