Quantifying uncertainty of taxonomic placement in DNA barcoding and metabarcoding

Abstract

O_LIA crucial step in the use of DNA markers for biodiversity surveys is the assignment of Linnaean taxonomies (species, genus, etc.) to sequence reads. This allows the use of all the information known based on the taxonomic names. Taxonomic placement of DNA barcoding sequences is inherently probabilistic because DNA sequences contain errors, because there is natural variation among sequences within a species, and because reference databases are incomplete and can have false annotations. However, most existing bioinformatics methods for taxonomic placement either exclude uncertainty, or quantify it using metrics other than probability.\nC_LIO_LIIn this paper we evaluate the performance of a recently proposed probabilistic taxonomic placement method PROTAX by applying it to both annotated reference sequence data as well as unknown environmental data. Our four case studies include contrasting taxonomic groups (fungi, bacteria, mammals, and insects), variation in the length and quality of the barcoding sequences (from individually Sanger-sequenced sequences to short Illumina reads), variation in the structures and sizes of the taxonomies (from 800 to 130 000 species), and variation in the completeness of the reference databases (representing 15% to 100% of the species).\nC_LIO_LIOur results demonstrate that PROTAX yields essentially unbiased assessment of probabilities of taxonomic placement, and thus that its quantification of species identification uncertainty is reliable. As expected, the accuracy of taxonomic placement increases with increasing coverage of taxonomic and reference sequence databases, and with increasing ratio of genetic variation among taxonomic levels over within taxonomic levels.\nC_LIO_LIOur results show that reliable species-level identification from environmental samples is still challenging, and thus neglecting identification uncertainty can lead to spurious inference. A key aim for future research is the completion and pruning of taxonomic and reference sequence databases, and making these two types of data compatible.\nC_LI