A universal method for species identification of mammals utilizing next generation sequencing for the analysis of DNA mixtures

PLoS One. 2013 Dec 16;8(12):e83761. doi: 10.1371/journal.pone.0083761. eCollection 2013.


Species identification can be interesting in a wide range of areas, for example, in forensic applications, food monitoring and in archeology. The vast majority of existing DNA typing methods developed for species determination, mainly focuses on a single species source. There are, however, many instances where all species from mixed sources need to be determined, even when the species in minority constitutes less than 1 % of the sample. The introduction of next generation sequencing opens new possibilities for such challenging samples. In this study we present a universal deep sequencing method using 454 GS Junior sequencing of a target on the mitochondrial gene 16S rRNA. The method was designed through phylogenetic analyses of DNA reference sequences from more than 300 mammal species. Experiments were performed on artificial species-species mixture samples in order to verify the method's robustness and its ability to detect all species within a mixture. The method was also tested on samples from authentic forensic casework. The results showed to be promising, discriminating over 99.9 % of mammal species and the ability to detect multiple donors within a mixture and also to detect minor components as low as 1 % of a mixed sample.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA Fingerprinting / methods
  • Forensic Genetics
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Mammals / classification*
  • Mammals / genetics*
  • Phylogeny
  • Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Sequence Analysis, DNA / methods
  • Species Specificity

Grant support

This work was supported by grants from the Swedish National Board of Forensic Medicine (RMVFoU 05:25 and RMVFoU 11:03). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.