DNA barcodes enable higher taxonomic assignments in the Acari.
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ABSTRACT:
SUBMITTER: Young MR
PROVIDER: S-EPMC8342613 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Detecting and documenting the occurrence of invasive species outside their native range requires tools to support their identification. This can be challenging for taxa with diverse life stages and/or problematic or unresolved morphological taxonomies. DNA barcoding provides a potent method for identifying invasive species, as it allows for species identification at all life stages, including fragmentary remains. It also provides an efficient interim taxonomic framework for quantifying cryptic genetic diversity by parsing barcode sequences into discontinuous haplogroup clusters (typical of reproductively isolated species) and labelling them with unique alphanumeric identifiers. Snakehead fishes are a diverse group of opportunistic predators endemic to Asia and Africa that may potentially pose significant threats as aquatic invasive species. At least three snakehead species (Channa argus, C. maculata, and C. marulius) are thought to have entered North America through the aquarium and live-food fish markets, and have established populations, yet their origins remain unclear. The objectives of this study were to assemble a library of DNA barcode sequences derived from expert identified reference specimens in order to determine the identity and aid invasion pathway analysis of the non-indigenous species found in North America using DNA barcodes. Sequences were obtained from 121 tissue samples representing 25 species and combined with public records from GenBank for a total of 36 putative species, which then partitioned into 49 discrete haplogroups. Multiple divergent clusters were observed within C. gachua, C. marulius, C. punctata and C. striata suggesting the potential presence of cryptic species diversity within these lineages. Our findings demonstrate that DNA barcoding is a valuable tool for species identification in challenging and under-studied taxonomic groups such as snakeheads, and provides a useful framework for inferring invasion pathway analysis.
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Project description:BackgroundIn metabarcoding analyses, the taxonomic assignment is crucial to place sequencing data in biological and ecological contexts. This fundamental step depends on a reference database, which should have a good taxonomic coverage to avoid unassigned sequences. However, this goal is rarely achieved in many geographic regions and for several taxonomic groups. On the other hand, more is not necessarily better, as sequences in reference databases belonging to taxonomic groups out of the studied region/environment context might lead to false assignments.MethodsWe investigated the effect of using several subsets of a cytochrome c oxidase subunit I (COI) reference database on taxonomic assignment. Published metabarcoding sequences from the Mediterranean Sea were assigned to taxa using COInr, which is a comprehensive, non-redundant and recent database of COI sequences obtained both from BOLD and NCBI, and two of its subsets: (i) all sequences except insects (COInr-WO-Insecta), which represent the overwhelming majority of COInr database, but are irrelevant for marine samples, and (ii) all sequences from taxonomic families present in the Mediterranean Sea (COInr-Med). Four different algorithms for taxonomic assignment were employed in parallel to evaluate differences in their output and data consistency.ResultsThe reduction of the database to more specific custom subsets increased the number of unassigned sequences. Nevertheless, since most of them were incorrectly assigned by the less specific databases, this is a positive outcome. Moreover, the taxonomic resolution (the lowest taxonomic level to which a sequence is attributed) of several sequences tended to increase when using customized databases. These findings clearly indicated the need for customized databases adapted to each study. However, the very high proportion of unassigned sequences points to the need to enrich the local database with new barcodes specifically obtained from the studied region and/or taxonomic group. Including novel local barcodes to the COI database proved to be very profitable: by adding only 116 new barcodes sequenced in our laboratory, thus increasing the reference database by only 0.04%, we were able to improve the resolution for ca. 0.6-1% of the Amplicon Sequence Variants (ASVs).
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Project description:Multiparametric imaging allows researchers to measure the expression of many biomarkers simultaneously, allowing detailed characterization of cell microenvironments. One such technique, CODEX, allows fluorescence imaging of >30 proteins in a single tissue section. In the commercial CODEX system, primary antibodies are conjugated to DNA barcodes. This modification can result in antibody dysfunction, and development of a custom antibody panel can be very costly and time consuming as trial and error of modified antibodies proceeds. To address these challenges, we developed novel tyramide-conjugated DNA barcodes that can be used with primary antibodies via peroxidase-conjugated secondary antibodies. This approach results in signal amplification and imaging without the need to conjugate primary antibodies. When combined with commercially available barcode-conjugated primary antibodies, we can very quickly develop working antibody panels. We also present methods to perform antibody staining using a commercially available automated tissue stainer and in situ hybridization imaging on a CODEX platform. Future work will include application of the combined tyramide-based and regular CODEX approach to image specific tumors with their immune cell infiltrates, including biomarkers that are currently difficult to image by regular CODEX.
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Project description:Each holotype specimen provides the only objective link to a particular Linnean binomen. Sequence information from them is increasingly valuable due to the growing usage of DNA barcodes in taxonomy. As type specimens are often old, it may only be possible to recover fragmentary sequence information from them. We tested the efficacy of short sequences from type specimens in the resolution of a challenging taxonomic puzzle: the Elachista dispunctella complex which includes 64 described species with minuscule morphological differences. We applied a multistep procedure to resolve the taxonomy of this species complex. First, we sequenced a large number of newly collected specimens and as many holotypes as possible. Second, we used all >400 bp examine species boundaries. We employed three unsupervised methods (BIN, ABGD, GMYC) with specified criteria on how to handle discordant results and examined diagnostic bases from each delineated putative species (operational taxonomic units, OTUs). Third, we evaluated the morphological characters of each OTU. Finally, we associated short barcodes from types with the delineated OTUs. In this step, we employed various supervised methods, including distance-based, tree-based and character-based. We recovered 658 bp barcode sequences from 194 of 215 fresh specimens and recovered an average of 141 bp from 33 of 42 holotypes. We observed strong congruence among all methods and good correspondence with morphology. We demonstrate potential pitfalls with tree-, distance- and character-based approaches when associating sequences of varied length. Our results suggest that sequences as short as 56 bp can often provide valuable taxonomic information. The results support significant taxonomic oversplitting of species in the Elachista dispunctella complex.
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Project description:DNA barcodes can facilitate identification of organisms especially when morphological characters are limited or unobservable. To what extent this potential is realized in specific groups of plants remains to be determined. Libraries of barcode sequences from well-studied authoritatively identified plants represented by herbarium voucher specimens are needed in order for DNA barcodes to serve their intended purpose, where this is possible, and to understand the reasons behind their failure to do so, when this occurs. We evaluated four loci, widely regarded as universal DNA barcodes for plants, for their utility in hawthorn species identification. Three plastid regions, matK, rbcLa and psbA-trnH, and the internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA discriminate only some of the species of Crataegus that can be recognized on the basis of their morphology etc. This is, in part, because in Rosaceae tribe Maleae most individual plastid loci yield relatively little taxonomic resolution and, in part, because the effects of allopolyploidization have not been eliminated by concerted evolution of the ITS regions. Although individual plastid markers provided generally poor resolution of taxonomic groups in Crataegus, a few species were notable exceptions. In contrast, analyses of concatenated sequences of the 3 plastid barcode loci plus 11 additional plastid loci gave a well-resolved maternal phylogeny. In the ITS2 tree, different individuals of some species formed groups with taxonomically unrelated species. This is a sign of lineage sorting due to incomplete concerted evolution in ITS2. Incongruence between the ITS2 and plastid trees is best explained by hybridization between different lineages within the genus. In aggregate, limited between-species variation in plastid loci, hybridization and a lack of concerted evolution in ITS2 all combine to limit the utility of standard barcoding markers in Crataegus. These results have implications for authentication of hawthorn materials in natural health products.
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Project description:Microhyla ornata, a species originally described from the southwest coast of India in 1841, was long reported to be wide-ranging throughout South, Southeast, and East Asia. Although the name M. ornata is restricted to populations from South Asia, the species is still considered to occur widely in India and its neighboring regions. To clarify the identity and geographical distribution of M. 'ornata', we performed DNA barcoding using a fragment of the mitochondrial 16S rRNA gene from 62 newly obtained samples. Our results show that this taxon is restricted to Peninsular India and Sri Lanka, whereas, populations from the other parts represent three different species - M. mukhlesuri, M. mymensinghensis, and M. nilphamariensis, creating new country records for India. Our work reemphasizes the benefits of DNA barcoding for rapidly identifying populations of widespread species and provides insights into the patterns of genetic differentiation in the M. 'ornata' species complex of South Asia.
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Project description:CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.
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Project description:Recent declines of insect populations at high rates have resulted in the need to develop a quick method to determine their diversity and to process massive data for the identification of species of highly diverse groups. A short sequence of DNA from COI is widely used for insect identification by comparing it against sequences of known species. Repositories of sequences are available online with tools that facilitate matching of the sequences of interest to a known individual. However, the performance of these tools can differ. Here we aim to assess the accuracy in identification of insect taxonomic categories from two repositories, BOLD Systems and GenBank. This was done by comparing the sequence matches between the taxonomist identification and the suggested identification from the platforms. We used 1,160 COI sequences representing eight orders of insects from Colombia. After the comparison, we reanalyzed the results from a representative subset of the data from the subfamily Scarabaeinae (Coleoptera). Overall, BOLD systems outperformed GenBank, and the performance of both engines differed by orders and other taxonomic categories (species, genus and family). Higher rates of accurate identification were obtained at family and genus levels. The accuracy was higher in BOLD for the order Coleoptera at family level, for Coleoptera and Lepidoptera at genus and species level. Other orders performed similarly in both repositories. Moreover, the Scarabaeinae subset showed that species were correctly identified only when BOLD match percentage was above 93.4% and a total of 85% of the samples were correctly assigned to a taxonomic category. These results accentuate the great potential of the identification engines to place insects accurately into their respective taxonomic categories based on DNA barcodes and highlight the reliability of BOLD Systems for insect identification in the absence of a large reference database for a highly diverse country.
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Project description:Taxonomic assignment of sequence reads is a challenging task in metagenomic data analysis, for which the present methods mainly use either composition- or homology-based approaches. Though the homology-based methods are more sensitive and accurate, they suffer primarily due to the time needed to generate the Blast alignments. We developed the MetaBin program and web server for better homology-based taxonomic assignments using an ORF-based approach. By implementing Blat as the faster alignment method in place of Blastx, the analysis time has been reduced by severalfold. It is benchmarked using both simulated and real metagenomic datasets, and can be used for both single and paired-end sequence reads of varying lengths (≥45 bp). To our knowledge, MetaBin is the only available program that can be used for the taxonomic binning of short reads (<100 bp) with high accuracy and high sensitivity using a homology-based approach. The MetaBin web server can be used to carry out the taxonomic analysis, by either submitting reads or Blastx output. It provides several options including construction of taxonomic trees, creation of a composition chart, functional analysis using COGs, and comparative analysis of multiple metagenomic datasets. MetaBin web server and a standalone version for high-throughput analysis are available freely at http://metabin.riken.jp/.
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Project description:Determining the taxonomic lineage of DNA sequences is an important step in metagenomic analysis. Short DNA fragments from next-generation sequencing projects and microbes that lack close relatives in reference sequenced genome databases pose significant problems to taxonomic attribution methods. Our new classification algorithm, RITA (Rapid Identification of Taxonomic Assignments), uses the agreement between composition and homology to accurately classify sequences as short as 50 nt in length by assigning them to different classification groups with varying degrees of confidence. RITA is much faster than the hybrid PhymmBL approach when comparable homology search algorithms are used, and achieves slightly better accuracy than PhymmBL on an artificial metagenome. RITA can also incorporate prior knowledge about taxonomic distributions to increase the accuracy of assignments in data sets with varying degrees of taxonomic novelty, and classified sequences with higher precision than the current best rank-flexible classifier. The accuracy on short reads can be increased by exploiting paired-end information, if available, which we demonstrate on a recently published bovine rumen data set. Finally, we develop a variant of RITA that incorporates accelerated homology search techniques, and generate predictions on a set of human gut metagenomes that were previously assigned to different 'enterotypes'. RITA is freely available in Web server and standalone versions.
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