RT Journal Article SR Electronic T1 A simple centrifugation protocol leads to a 55-fold mitochondrial DNA enrichment and paves the way for future mitogenomic research JF bioRxiv FD Cold Spring Harbor Laboratory SP 106583 DO 10.1101/106583 A1 Jan Niklas Macher A1 Vera Zizka A1 Alexander Martin Weigand A1 Florian Leese YR 2017 UL http://biorxiv.org/content/early/2017/02/10/106583.abstract AB 1. DNA (meta)barcoding is increasingly used to study and monitor biodiversity and available for standardised assessments. However, it suffers from PCR bias which can lead to the loss of taxonomic groups. PCR-free techniques such as metagenomics are therefore thought to be more suited for biodiversity assessments but are currently limited by incomplete reference libraries.2. The technique of ‘mitogenome-skimming’ or ‘mitogenomics’, where complete mitochondrial genomes are sequenced, is ideal to bridge the techniques of (meta)barcoding and metagenomics. 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 a simple centrifugation protocol that leads to a 55-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 with a minimum coverage of 2417-fold. In addition, we ‘skimmed’ the whole mitochondrial genome of the acanthocephalan endoparasite Pomphorhynchus laevis (444-fold coverage) obviously being present in G. roeselii.4. Correlation between body mass of specimens in each community and generated sequence number was moderate in samples enriched for mitochondria. Potential problems arise with species demonstrating a high weight, but produce less than expected mitochondrial reads (e.g. shelled organisms). The developed protocol will greatly 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 enrichment for mitochondria.