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Background One of the main challenges in metagenomics is the identification of microorganisms in clinical and environmental samples. While an extensive and heterogeneous set of computational tools is available to classify microorganisms using whole-genome shotgun sequencing data, comprehensive comparisons of these methods are limited. Results In this study, we use the largest-to-date set of laboratory-generated and simulated controls across 846 species to evaluate the performance of 11 metagenomic classifiers. Tools were characterized on the basis of their ability to identify taxa at the genus, species, and strain levels, quantify relative abundances of taxa, and classify individual reads to the species level. Strikingly, the number of species identified by the 11 tools can differ by over three orders of magnitude on the same datasets. Various strategies can ameliorate taxonomic misclassification, including abundance filtering, ensemble approaches, and tool intersection. Nevertheless, these strategies were often insufficient to completely eliminate false positives from environmental samples, which are especially important where they concern medically relevant species. Overall, pairing tools with different classification strategies (k-mer, alignment, marker) can combine their respective advantages. Conclusions This study provides positive and negative controls, titrated standards, and a guide for selecting tools for metagenomic analyses by comparing ranges of precision, accuracy, and recall. We show that proper experimental design and analysis parameters can reduce false positives, provide greater resolution of species in complex metagenomic samples, and improve the interpretation of results.

Environmental DNA (eDNA) techniques are gaining attention as cost-effective, non-invasive strategies for acquiring information on fish and other aquatic organisms from water samples. Currently, eDNA approaches are used to detect specific fish species and determine fish community diversity. Various protocols used with eDNA methods for aquatic organism detection have been reported in different eDNA studies, but there are no general recommendations for fish detection. Herein, we reviewed 168 papers to supplement and highlight the key criteria for each step of eDNA technology in fish detection and provide general suggestions for eliminating detection errors. Although there is no unified recommendation for the application of diverse eDNA in detecting fish species, in most cases, 1 or 2 L surface water collection and eDNA capture on 0.7-μm glass fiber filters followed by extraction with a DNeasy Blood and Tissue Kit or PowerWater DNA Isolation Kit are useful for obtaining high-quality eDNA. Subsequently, species-specific quantitative polymerase chain reaction (qPCR) assays based on mitochondrial cytochrome b gene markers or eDNA metabarcoding based on both 12S and 16S rRNA markers via high-throughput sequencing can effectively detect target DNA or estimate species richness. Furthermore, detection errors can be minimized by mitigating contamination, negative control, PCR replication, and using multiple genetic markers. Our aim is to provide a useful strategy for fish eDNA technology that can be applied by researchers, advisors, and managers.

Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines.

DNA barcoding using a fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (COI) has proven to be successful for species-level identification in many animal groups. However, most studies have been focused on relatively small datasets or on large datasets of taxonomically high-ranked groups. We explore the quality of DNA barcodes to delimit species in the diverse chironomid genus Tanytarsus (Diptera: Chironomidae) by using different analytical tools. The genus Tanytarsus is the most species-rich taxon of tribe Tanytarsini (Diptera: Chironomidae) with more than 400 species worldwide, some of which can be notoriously difficult to identify to species-level using morphology. Our dataset, based on sequences generated from own material and publicly available data in BOLD, consist of 2790 DNA barcodes with a fragment length of at least 500 base pairs. A neighbor joining tree of this dataset comprises 131 well separated clusters representing 121 morphological species of Tanytarsus: 77 named, 16 unnamed and 28 unidentified theoretical species. For our geographically widespread dataset, DNA barcodes unambiguously discriminate 94.6% of the Tanytarsus species recognized through prior morphological study. Deep intraspecific divergences exist in some species complexes, and need further taxonomic studies using appropriate nuclear markers as well as morphological and ecological data to be resolved. The DNA barcodes cluster into 120-242 molecular operational taxonomic units (OTUs) depending on whether Objective Clustering, Automatic Barcode Gap Discovery (ABGD), Generalized Mixed Yule Coalescent model (GMYC), Poisson Tree Process (PTP), subjective evaluation of the neighbor joining tree or Barcode Index Numbers (BINs) are used. We suggest that a 4-5% threshold is appropriate to delineate species of Tanytarsus non-biting midges.

The transfer of ancestral plastid genomes into mitochondrial genomes to generate mitochondrial plastid DNA (MTPT) is known to occur in plants, but its impacts on mitochondrial genome complexity and the potential for causing a false-positive DNA barcoding paradox have been underestimated. Here, we assembled the organelle genomes of Cynanchum wilfordii and C. auriculatum , which are indigenous medicinal herbs in Korea and China, respectively. In both species, it is estimated that 35% of the ancestral plastid genomes were transferred to mitochondrial genomes over the past 10 million years and remain conserved in these genomes. Some plastid barcoding markers co-amplified the conserved MTPTs and caused a barcoding paradox, resulting in mis-authentication of botanical ingredients and/or taxonomic mis-positioning. We identified dynamic and lineage-specific MTPTs that have contributed to mitochondrial genome complexity and might cause a putative barcoding paradox across 81 plant species. We suggest that a DNA barcoding guidelines should be developed involving the use of multiple markers to help regulate economically motivated adulteration.

Several recent estimates of global biodiversity have concluded that the total number of species on Earth lies near the lower end of the wide range touted in previous decades. However, none of these recent estimates formally explore the real \"elephant in the room\", namely, what proportion of species are taxonomically invisible to conventional assessments, and thus, as undiagnosed cryptic species, remain uncountable until revealed by multi-gene molecular assessments. Here we explore the significance and extent of so-called \"hyper-cryptic\" species complexes, using the Australian freshwater fish Galaxias olidus as a proxy for any organism whose taxonomy ought to be largely finalized when compared to those in littlestudied or morphologically undifferentiated groups. Our comprehensive allozyme (838 fish for 54 putative loci), mtDNA (557 fish for 605 bp of cytb), and morphological (1963-3389 vouchers for 17-58 characters) assessment of this species across its broad geographic range revealed a 1500% increase in species-level biodiversity, and suggested that additional taxa may remain undiscovered. Importantly, while all 15 candidate species were morphologically diagnosable a posteriori from one another, single-gene DNA barcoding proved largely unsuccessful as an a priori method for species identification. These results lead us to draw two strong inferences of relevance to estimates of global biodiversity. First, hyper-cryptic complexes are likely to be common in many organismal groups. Second, no assessment of species numbers can be considered \"best practice\" in the molecular age unless it explicitly includes estimates of the extent of cryptic and hyper-cryptic biodiversity.

Background As the increased consumption of ready-to-eat meat alternatives is a fairly recent trend, little is known about the composition and dynamics of the microbiota present on such products. Such information is nonetheless valuable in view of spoilage and food safety prevention. Even though refrigeration and modified-atmosphere-packaging (MAP) can extend the shelf-life period, microbial spoilage can still occur in these products. In the present study, the microbiota of a vegetarian alternative to poultry-based charcuterie was investigated during storage, contrasting the use of a culture-dependent method to a culture-independent metagenetic method. Results The former revealed that lactic acid bacteria (LAB) were the most abundant microbial group, specifically at the end of the shelf-life period, whereby Latilactobacillus sakei was the most abundant species. Metabarcoding analysis, in contrast, revealed that DNA of Xanthomonas was most prominently present, which likely was an artifact due to the presence of xanthan gum as an ingredient, followed by Streptococcus and Weissella . Conclusions Taken together, these results indicated that Lb. sakei was likely the most prominent specific spoilage organisms (SSO) and, additionally, that the use of metagenetic analysis needs to be interpreted with care in this specific type of product. In order to improve the performance of metagenetics in food samples with a high DNA matrix but a low bacterial DNA load, selective depletion techniques for matrix DNA could be explored.

Rodentia is the most diverse order among mammals, with more than 2,000 species currently described. Most of the time, species assignation is so difficult based on morphological data solely that identifying rodents at the specific level corresponds to a real challenge. In this study, we compared the applicability of 100 bp mini-barcodes from cytochrome b and cytochrome c oxidase 1 genes to enable rodent species identification. Based on GenBank sequence datasets of 115 rodent species, a 136 bp fragment of cytochrome b was selected as the most discriminatory mini-barcode, and rodent universal primers surrounding this fragment were designed. The efficacy of this new molecular tool was assessed on 946 samples including rodent tissues, feces, museum samples and feces/pellets from predators known to ingest rodents. Utilizing next-generation sequencing technologies able to sequence mixes of DNA, 1,140 amplicons were tagged, multiplexed and sequenced together in one single 454 GS-FLX run. Our method was initially validated on a reference sample set including 265 clearly identified rodent tissues, corresponding to 103 different species. Following validation, 85.6% of 555 rodent samples from Europe, Asia and Africa whose species identity was unknown were able to be identified using the BLASTN program and GenBank reference sequences. In addition, our method proved effective even on degraded rodent DNA samples: 91.8% and 75.9% of samples from feces and museum specimens respectively were correctly identified. Finally, we succeeded in determining the diet of 66.7% of the investigated carnivores from their feces and 81.8% of owls from their pellets. Non-rodent species were also identified, suggesting that our method is sensitive enough to investigate complete predator diets. This study demonstrates how this molecular identification method combined with high-throughput sequencing can open new realms of possibilities in achieving fast, accurate and inexpensive species identification.

Morphological study of 1,795 spiders from sites across Pakistan placed these specimens in 27 families and 202 putative species. COI sequences >400 bp recovered from 1,782 specimens were analyzed using neighbor-joining trees, Bayesian inference, barcode gap, and Barcode Index Numbers (BINs). Specimens of 109 morphological species were assigned to 123 BINs with ten species showing BIN splits, while 93 interim species included representatives of 98 BINs. Maximum conspecific divergences ranged from 0-5.3% while congeneric distances varied from 2.8-23.2%. Excepting one species pair (Oxyopes azhari-Oxyopes oryzae), the maximum intraspecific distance was always less than the nearest-neighbor (NN) distance. Intraspecific divergence values were not significantly correlated with geographic distance. Most (75%) BINs detected in this study were new to science, while those shared with other nations mainly derived from India. The discovery of many new, potentially endemic species and the low level of BIN overlap with other nations highlight the importance of constructing regional DNA barcode reference libraries.

The increasing availability of reference libraries of DNA barcodes (RLDB) offers the opportunity to the screen the level of consistency in DNA barcode data among libraries, in order to detect possible disagreements generated from taxonomic uncertainty or operational shortcomings. We propose a ranking system to attribute a confidence level to species identifications associated with DNA barcode records from a RLDB. Here we apply the proposed ranking system to a newly generated RLDB for marine fish of Portugal. Specimens (n = 659) representing 102 marine fish species were collected along the continental shelf of Portugal, morphologically identified and archived in a museum collection. Samples were sequenced at the barcode region of the cytochrome oxidase subunit I gene (COI-5P). Resultant DNA barcodes had average intra-specific and inter-specific Kimura-2-parameter distances (0.32% and 8.84%, respectively) within the range usually observed for marine fishes. All specimens were ranked in five different levels (A-E), according to the reliability of the match between their species identification and the respective diagnostic DNA barcodes. Grades A to E were attributed upon submission of individual specimen sequences to BOLD-IDS and inspection of the clustering pattern in the NJ tree generated. Overall, our study resulted in 73.5% of unambiguous species IDs (grade A), 7.8% taxonomically congruent barcode clusters within our dataset, but awaiting external confirmation (grade B), and 18.7% of species identifications with lower levels of reliability (grades C/E). We highlight the importance of implementing a system to rank barcode records in RLDB, in order to flag taxa in need of taxonomic revision, or reduce ambiguities of discordant data. With increasing DNA barcode records publicly available, this cross-validation system would provide a metric of relative accuracy of barcodes, while enabling the continuous revision and annotation required in taxonomic work.