Testing the Reliability of Genetic Methods of Species Identification via Simulation

Abstract

Although genetic methods of species identification, especially DNA barcoding, are strongly debated, tests of these methods have been restricted to a few empirical cases for pragmatic reasons. Here we use simulation to test the performance of methods based on sequence comparison (BLAST and genetic distance) and tree topology over a wide range of evolutionary scenarios. Sequences were simulated on a range of gene trees spanning almost three orders of magnitude in tree depth and in coalescent depth; that is, deep or shallow trees with deep or shallow coalescences. When the query's conspecific sequences were included in the reference alignment, the rate of positive identification was related to the degree to which different species were genetically differentiated. The BLAST, distance, and liberal tree-based methods returned higher rates of correct identification than did the strict tree-based requirement that the query was within, but not sister to, a single-species clade. Under this more conservative approach, ambiguous outcomes occurred in inverse proportion to the number of reference sequences per species. When the query's conspecific sequences were not in the reference alignment, only the strict tree-based approach was relatively immune to making false-positive identifications. Thresholds affected the rates at which false-positive identifications were made when the query's species was unrepresented in the reference alignment but did not otherwise influence outcomes. A conservative approach using the strict tree-based method should be used initially in large-scale identification systems, with effort made to maximize sequence sampling within species. Once the genetic variation within a taxonomic group is well characterized and the taxonomy resolved, then the choice of method used should be dictated by considerations of computational efficiency. The requirement for extensive genetic sampling may render these techniques inappropriate in some circumstances.

Utility of DNA barcoding to identify rare endemic vascular plant species in Trinidad

“…There is no single optimal method to determine the resolving power of DNA barcodes for all taxa (Austerlitz et al., 2009; Casiraghi et al., 2010; Collins & Cruickshank, 2013; Meyer & Paulay, 2005; Moritz & Cicero, 2004; Ross et al., 2008). Different approaches exist for matching an unknown query sequence with sequences in a reference database or library and tend to be based on ad hoc criteria which may include the frequency of the highest hits, percentage sequence similarity, bootstrapping, BLAST scores or tree‐based clustering assessment (Kress et al., 2009; Wilson et al.…”

“…In this study, NJ trees were used to establish clustering of query sequences into correct genus‐specific groups with strong bootstrap support and were not used to infer phylogeny. Poor resolution in tree topologies with low bootstrap scores and polytomies obtained for rbcL sequences were obtained which may be due to inadequate low genetic distances for most species (Hebert et al., 2003; Hollingsworth et al., 2016; Kress et al., 2009; Ross et al., 2008; Wilson et al. 2011).…”

“…A “correct” assignment was then checked with the morphology‐based identification and if there was concordance, the assignment was considered to be TRUE, or FALSE if there was disagreement with the morphology‐based identification (Ross, Murugan, & Li, 2008; Wilson et al., 2011). An “ambiguous” assignment was concluded where the true taxon based on morphological descriptors was not represented in the reference dataset for that barcode which meant that the library was incomplete with inadequate coverage for that specific taxon (Ross et al., 2008; Wilson et al., 2011). …”

Five simple guidelines for establishing basic authenticity and reliability of newly generated fungal ITS sequences

“…Such simple queries permit the user to examine and establish whether the expected species are present among the available ITS sequences in the database. Ross et al (2008) and Ovaskainen et al (2010) provide interesting statistics on the performance of BLAST under varying conditions, including incomplete database coverage.…”

The phylogenetic analysis of Dalbergia (Fabaceae: Papilionaceae) based on different DNA barcodes

“…The correct identification rates of all barcodes in ASB were rapidly decreased comparing to BCM, except rbcL (increased from 34.4% to 43.9%). Singletons are also a source of incorrect identification due to the absence of other conspecific reference sequences in the dataset without matching (Ross et al 2008). Lahaye et al (2008) showed that matK and rbcL barcoding cannot yield a barcode gap in the closely related plant species.…”