Fishing for Mitochondrial DNA in The Egyptian Sacred Ibis Mummies

Abstract

The long-term preservation of DNA requires a number of optimal conditions, including consistent exposure to cool, dry, and dark environments. As a result, the successful recovery of ancient DNA from material from warmer climates such as those in Egypt has often been met with scepticism. Egypt has an abundance of ancient mummified animals and humans, whose genetic analyses would offer important insights into ancient cultural practices. To date, the retrieval of complete genomes from ancient Egyptian remains of humans or other animals has been largely unsuccessful. To test for the presence of even short DNA sequences in Egyptian material, we performed second-generation shotgun sequencing of DNA libraries constructed from ancient Sacred Ibis mummies. Since most of the resulting Illumina libraries were shown to contain extremely low levels (less than 0.06%) of endogenous mitochondrial DNA, we aimed to enrich these samples using targeted in-solution hybridisation methods. Using biotinylated RNA baits designed to Sacred Ibis complete mitochondrial sequences, we trialled a number of conditions and parameters and achieved up to 4705-fold enrichment. We also found that a combination of hybridisation temperature and the use of the polymerase KAPA HiFi significantly increased both the efficiency of targeted hybridisation and post-hybridisation amplification respectively. Furthermore, improved enrichment was accompanied with only minor increases in clonality. Our method enabled us to reconstruct the first complete mitochondrial genomes from ancient Egyptian sub-fossil material.