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1. A molecular assessment of species diversity in Tympanopleura and Ageneiosus catfishes (Auchenipteridae: Siluriformes)

   https://doi.org/10.1111/jfb.14173  

   Hashimoto, Shizuka ; Py‐Daniel, Lúcia H. Rapp ; Batista, Jacqueline S. ( November 2019 , Journal of Fish Biology)

   In order to test the congruence of genetic data to the morphologically defined Neotropical catfish generaTympanopleuraandAgeneiosusand explore species diversity, we generated 17 DNA barcodes from five of six species ofTympanopleuraand 12 of 13 species ofAgeneiosus. To discriminate limits between species, an automatic barcode gap discovery (ABGD), a generalised mixed yule‐coalescent model (GYMC) and fixed distance thresholds Kimura two‐parameter (K2P; 3%) were used to discriminate putative species limits from the DNA barcodes. The ABGD, GMYC and K2P methods agreed by each generating 13 clusters: six inTympanopleura(five nominal plus one undescribed species) and seven inAgeneiosus. These clusters corresponded broadly to the described species, except in the case of theAgeneiosus ucayalensisgroup (A. akamai,A. dentatus,A. intrusus,A. ucayalensis,A. uranophthalmusandA. vittatus). Haplotype sharing and low divergences may have prevented molecular methods from distinguishing these species. We hypothesise that this is the result of a recent radiation of a sympatric species group distributed throughout the Amazon Basin. One putative new species ofTympanopleurawas also supported by the molecular data. These results taken together highlight the utility of molecular methods such as DNA barcoding in understanding patterns of diversification across large geographic areas and in recognising overlooked diversity.

    

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2. Most soil and litter arthropods are unidentifiable based on current DNA barcode reference libraries

   https://doi.org/10.1093/cz/zoad051  

   Recuero, Ernesto ; Etzler, Frank E. ; Caterino, Michael S. ; Jia, ed., Zhi-Yun ( November 2023 , Current Zoology)

   We are far from knowing all species living on the planet. Understanding biodiversity is demanding and requires time and expertise. Most groups are understudied given problems of identifying and delimiting species. DNA barcoding emerged to overcome some of the difficulties in identifying species. Its limitations derive from incomplete taxonomic knowledge and the lack of comprehensive DNA barcode libraries for so many taxonomic groups. Here, we evaluate how useful barcoding is for identifying arthropods from highly diverse leaf litter communities in the southern Appalachian Mountains (USA). We used 3 reference databases and several automated classification methods on a data set including several arthropod groups. Acari, Araneae, Collembola, Coleoptera, Diptera, and Hymenoptera were well represented, showing different performances across methods and databases. Spiders performed the best, with correct identification rates to species and genus levels of ~50% across databases. Springtails performed poorly, no barcodes were identified to species or genus. Other groups showed poor to mediocre performance, from around 3% (mites) to 20% (beetles) correctly identified barcodes to species, but also with some false identifications. In general, BOLD-based identification offered the best identification results but, in all cases except spiders, performance is poor, with less than a fifth of specimens correctly identified to genus or species. Our results indicate that the soil arthropod fauna is still insufficiently documented, with many species unrepresented in DNA barcode libraries. More effort toward integrative taxonomic characterization is needed to complete our reference libraries before we can rely on DNA barcoding as a universally applicable identification method.

    

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3. Best Practices in Designing, Sequencing, and Identifying Random DNA Barcodes

   https://doi.org/10.1007/s00239-022-10083-z  

   Johnson, Milo S. ; Venkataram, Sandeep ; Kryazhimskiy, Sergey ( January 2023 , Journal of Molecular Evolution)

   Random DNA barcodes are a versatile tool for tracking cell lineages, with applications ranging from development to cancer to evolution. Here, we review and critically evaluate barcode designs as well as methods of barcode sequencing and initial processing of barcode data. We first demonstrate how various barcode design decisions affect data quality and propose a new design that balances all considerations that we are currently aware of. We then discuss various options for the preparation of barcode sequencing libraries, including inline indices and Unique Molecular Identifiers (UMIs). Finally, we test the performance of several established and new bioinformatic pipelines for the extraction of barcodes from raw sequencing reads and for error correction. We find that both alignment and regular expression-based approaches work well for barcode extraction, and that error-correction pipelines designed specifically for barcode data are superior to generic ones. Overall, this review will help researchers to approach their barcoding experiments in a deliberate and systematic way.

    

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4. A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

   https://doi.org/10.3390/agronomy11071274  

   Chen, Ko-Hsuan ; Longley, Reid ; Bonito, Gregory ; Liao, Hui-Ling ( July 2021 , Agronomy)

   null (Ed.)

   High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest. 

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5. Isotopically Encoded Nanotags for Multiplexed Ion Beam Imaging

   https://doi.org/10.1002/admt.202000098  

   Harmsen, Stefan ; Coskun, Ahmet F. ; Ganesh, Shambavi ; Nolan, Garry P. ; Gambhir, Sanjiv S. ( May 2020 , Advanced Materials Technologies)

   High‐dimensional profiling of markers and analytes using approaches, such as barcoded fluorescent imaging with repeated labeling and mass cytometry has allowed visualization of biological processes at the single‐cell level. To address limitations of sensitivity and mass‐channel capacity, a nanobarcoding platform is developed for multiplexed ion beam imaging (MIBI) using secondary ion beam spectrometry that utilizes fabricated isotopically encoded nanotags. Use of combinatorial isotope distributions in 100 nm sized nanotags expands the labeling palette to overcome the spectral bounds of mass channels. As a proof‐of‐principle, a four‐digit (i.e., 0001–1111) barcoding scheme is demonstrated to detect 16 variants of²H,¹⁹F,^79/81Br, and¹²⁷I elemental barcode sets that are encoded in silica nanoparticle matrices. A computational debarcoding method and an automated machine learning analysis approach are developed to extract barcodes for accurate quantification of spatial nanotag distributions in large ion beam imaging areas up to 0.6 mm². Isotopically encoded nanotags should boost the performance of mass imaging platforms, such as MIBI and other elemental‐based bioimaging approaches.

    

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