MTaxi: A comparative tool for taxon identification of ultra low coverage ancient genomes

Abstract

A major challenge in zooarchaeology is to morphologically distinguish closely related species' remains, especially using small bone fragments. Shotgun sequencing aDNA from archeological remains and comparative alignment to the candidate species' reference genomes will only apply when reference nuclear genomes of comparable quality are available, and may still fail when coverages are low. Here, we propose an alternative method, MTaxi, that uses highly accessible mitochondrial DNA (mtDNA) to distinguish between pairs of closely related species from ancient DNA sequences. MTaxi utilises mtDNA transversion-type substitutions between pairs of candidate species, assigns reads to either species, and performs a binomial test to determine the sample taxon. We tested MTaxi on sheep/goat and horse/donkey data, between which zooarchaeological classification can be challenging in ways that epitomise our case. The method performed efficiently on simulated ancient genomes down to 0.3x mitochondrial coverage for both sheep/goat and horse/donkey, with no false positives. Trials on n=18 ancient sheep/goat samples and n=10 horse/donkey samples of known species identity also yielded 100% accuracy. Overall, MTaxi provides a straightforward approach to classify closely related species that are difficult to distinguish through zooarchaeological methods using low coverage aDNA data, especially when similar quality reference genomes are unavailable. MTaxi is freely available at https://github.com/goztag/MTaxi.

Keywords: ancient DNA; bioinformatics; genomics; mtDNA; zooarcheology.

Figure 1.. Overview of the MTaxi pipeline.

Flowchart and representations of the steps to determine the sample taxon. Here sheep and goat stand for the candidate species pair, but MTaxi can be applied to any pair of species where mitochondrial DNA reference sequences are available. Target sites represent mismatches (candidate substitutions) between the reference genomes, restricted to transversions. Blue coloured alleles represent transitions, red coloured alleles represent transversions. Reads are assigned to either taxon based on target sites. Purple coloured alleles represent sheep alleles, orange coloured alleles represent goat alleles. Reads may be assigned to the wrong taxon due to homoplastic mutations, technical error, or incomplete lineage sorting.

Figure 2.. Distribution of target sites along reference mitochondrial genomes.

The figure shows the position of target sites along ( a) sheep and ( b) goat ( c) horse ( d) donkey reference mitochondrial genomes. The sites represent transversion-type substitutions (n=197 for sheep and goat, and n=117 for horse and donkey). The figure was generated through CGview .

Figure 3.. Results of the method applied to simulated ancient genomes at different coverages.

Binomial test p-values for comparing the proportions of reads assigned to ( a) sheep versus goat, and ( b) and horse versus donkey. For sheep and goat, results are based on transversion substitutions, obtained through the default approach. n refers to the number of simulated genomes in each case (100 for each species in a pair). The height of the blue bar represents the number of simulated goat/donkey and sheep/horse genomes identified correctly with p<0.05, and the height of the red bar represents the number of unidentified cases. No cases were misidentified. The height of the grey bar represents the trials that did not contain any reads aligned to target sites, and thus could not be evaluated.

Figure 4.. Whole Genome Comparative Alignment Results.

Total number of bases aligned to sheep versus goat reference genomes (left panels) and the mismatch proportions (right panels) for whole genome ancient sheep (upper panels) and goat (lower panels) samples.