Instances of erroneous DNA barcoding of metazoan invertebrates: Are universal cox1 gene primers too “universal”?

Abstract

The cytochrome c oxidase subunit I (cox1) gene is the main mitochondrial molecular marker playing a pivotal role in phylogenetic research and is a crucial barcode sequence. Folmer’s “universal” primers designed to amplify this gene in metazoan invertebrates allowed quick and easy barcode and phylogenetic analysis. On the other hand, the increase in the number of studies on barcoding leads to more frequent publishing of incorrect sequences, due to amplification of non-target taxa, and insufficient analysis of the obtained sequences. Consequently, some sequences deposited in genetic databases are incorrectly described as obtained from invertebrates, while being in fact bacterial sequences. In our study, in which we used Folmer’s primers to amplify COI sequences of the crustacean fairy shrimp Branchipus schaefferi (Fischer 1834), we also obtained COI sequences of microbial contaminants from Aeromonas sp. However, when we searched the GenBank database for sequences closely matching these contaminations we found entries described as representatives of Gastrotricha and Mollusca. When these entries were compared with other sequences bearing the same names in the database, the genetic distance between the incorrect and correct sequences amplified from the same species was c.a. 65%. Although the responsibility for the correct molecular identification of species rests on researchers, the errors found in already published sequences data have not been re-evaluated so far. On the basis of the standard sampling technique we have estimated with 95% probability that the chances of finding incorrectly described metazoan sequences in the GenBank depend on the systematic group, and variety from less than 1% (Mollusca and Arthropoda) up to 6.9% (Gastrotricha). Consequently, the increasing popularity of DNA barcoding and metabarcoding analysis may lead to overestimation of species diversity. Finally, the study also discusses the sources of the problems with amplification of non-target sequences.

Why the COI barcode should be the community DNA metabarcode for the metazoa

“…Regarding other public databases (e.g. GenBank), effort is required to identify sequences with incorrect taxonomic assignment to avoid their use as reference data (Mioduchowska et al., ). (b) Development and validation of detailed and standardizable methods for fieldwork and extraction of the target specimens from their habitat matrix (water, soil, sediment, etc.)…”

“…Hausmann et al., ), stronger linkage is forged between traditional taxonomic systems and reference sequences. Barcode campaigns that employ rigorous taxonomic identification of voucher specimens also provide a necessary step forward to identify database sequences that have been incorrectly assigned taxonomically, as it has been shown to occur in the genbank database (Mioduchowska, Jan, Gołdyn, Kur, & Sell, ).…”

BOLD and GenBank revisited – Do identification errors arise in the lab or in the sequence libraries?

“…However, examination of the loan records at the USNM revealed that the loan of material to Meiklejohn et al contained multiple specimens which were not included in their analyses or mentioned in their article-and among them, two were Strepsiptera (USNM ENT 01248370 and 01248357). Cross-contamination is a well-recognized risk when working with museum specimens so it is standard practice to check for its occurrence [13,14]. While Meiklejohn et al [10] exercised some precautions in their laboratory protocols such as the incorporation of negative controls in PCR, there was no evidence that they considered the possibility that some of their DNA sequences derived from non-target taxa.…”

Gap-analysis of DNA barcoding in ERMS reference libraries for ascidians and cnidarians

“…Yet, being a public database, it is inevitable that BOLD, as any other similar curation tool, might accrue erroneous data, sometimes signi cantly [2,6]. Taxonomic misidenti cations and/or taxonomic conundrums, cryptic species complexes, delimiting cryptic species, technical faults, such as de cient DNA extraction, PCR-based errors and foreign DNA contaminations, including bacterial sequences, especially COI sequences, are just some of the causes that may unavoidably generate erroneous data and inaccurate sequences [2,[6][7][8][9][10]. The above di culties may affect dramatically the accuracy of barcoding.…”