Metabarcoding of environmental DNA (eDNA) and DNA extracted from bulk specimen samples is a powerful tool in studies of biodiversity, diet and ecological interactions as its inherent labelling of ...amplicons allows sequencing of taxonomically informative genetic markers from many samples in parallel. However, the occurrence of so‐called ‘tag‐jumps’ can cause incorrect assignment of sequences to samples and artificially inflate diversity. Two steps during library preparation of pools of 5ʹ nucleotide‐tagged amplicons have been suggested to cause tag‐jumps: (a) T4 DNA polymerase blunt‐ending in the end‐repair step and (b) postligation PCR amplification of amplicon libraries. The discovery of tag‐jumps has led to recommendations to only carry out metabarcoding PCR amplifications with primers carrying twin‐tags to ensure that tag‐jumps cannot result in false assignments of sequences to samples. As this increases both cost and workload, a metabarcoding library preparation protocol which circumvents the two steps that causes tag‐jumps is needed. Here, we demonstrate Tagsteady, a PCR‐free metabarcoding Illumina library preparation protocol for pools of nucleotide‐tagged amplicons that enables efficient and cost‐effective generation of metabarcoding data with virtually no tag‐jumps. We use pools of twin‐tagged amplicons to investigate the effect of T4 DNA polymerase blunt‐ending and postligation PCR on the occurrence of tag‐jumps and demonstrate that both blunt‐ending and postligation PCR, alone or together, can result in detrimental amounts of tag‐jumps (here, up to ca. 49% of total sequences), while leaving both steps out (the Tagsteady protocol) results in amounts of sequences carrying new combinations of used tags (tag‐jumps) comparable to background contamination.
The evolutionary history of the wolf-like canids of the genus Canis has been heavily debated, especially regarding the number of distinct species and their relationships at the population and species ...level 1–6. We assembled a dataset of 48 resequenced genomes spanning all members of the genus Canis except the black-backed and side-striped jackals, encompassing the global diversity of seven extant canid lineages. This includes eight new genomes, including the first resequenced Ethiopian wolf (Canis simensis), one dhole (Cuon alpinus), two East African hunting dogs (Lycaon pictus), two Eurasian golden jackals (Canis aureus), and two Middle Eastern gray wolves (Canis lupus). The relationships between the Ethiopian wolf, African golden wolf, and golden jackal were resolved. We highlight the role of interspecific hybridization in the evolution of this charismatic group. Specifically, we find gene flow between the ancestors of the dhole and African hunting dog and admixture between the gray wolf, coyote (Canis latrans), golden jackal, and African golden wolf. Additionally, we report gene flow from gray and Ethiopian wolves to the African golden wolf, suggesting that the African golden wolf originated through hybridization between these species. Finally, we hypothesize that coyotes and gray wolves carry genetic material derived from a “ghost” basal canid lineage.
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•Extensive gene flow in the genus Canis, especially among the crown group•Genetic contribution from an unknown canid into the ancestor of the gray wolf and coyote•The African golden wolf possibly a hybrid species, from the gray wolf and Ethiopian wolf•Possible ancient admixture between the dhole and African hunting dog
Gopalakrishnan et al. present evidence of pervasive gene flow among species of the genus Canis. In addition to previously known admixture events, they find evidence of gene flow from a “ghost” canid, related to the dhole, into the ancestor of the gray wolf and coyote. Further, they suggest that the African golden wolf is a species of hybrid origin.
Single‐tube library preparation for degraded DNA Carøe, Christian; Gopalakrishnan, Shyam; Vinner, Lasse ...
Methods in ecology and evolution,
February 2018, Volume:
9, Issue:
2
Journal Article
Peer reviewed
Open access
In recent years, massive parallel sequencing has revolutionised the study of degraded DNA, thus enabling the field of ancient DNA to evolve into that of paleogenomics. Despite these advances, the ...recovery and sequencing of degraded DNA remains challenging due to limitations in the manipulation of chemically damaged and highly fragmented DNA molecules. In particular, the enzymatic reactions and DNA purification steps during library preparation can result in DNA template loss and sequencing biases, affecting downstream analyses. The development of library preparation methods that circumvent these obstacles and enable higher throughput are therefore of interest to researchers working with degraded DNA.
In this study, we compare four Illumina library preparation protocols, including two “single‐tube” methods developed for this study with the explicit aim of improving data quality and reducing preparation time and expenses. The methods are tested on grey wolf (Canis lupus) museum specimens.
We found single‐tube protocols increase library complexity, yield more reads that map uniquely to the reference genome, reduce processing time, and may decrease laboratory costs by 90%.
Given the advantages of single‐tube library preparations, we anticipate these methods will be of considerable interest to the growing field of paleogenomics and other applications investigating degraded DNA.
Biodiversity monitoring projects using environmental DNA techniques are becoming increasingly widespread. However, these techniques depend heavily on the quality and richness of the available DNA ...reference database against which the DNA sequences are queried. To create a comprehensive DNA sequence database for future DNA‐based biodiversity assessments in Denmark, a national DNA reference database, DNAmark, was established, which contains organellar and/or nuclear reference data from vouchered museum species of plants, animals, and fungi from Denmark. Here, we present full or partial mitochondrial genomes of 182 Danish vertebrate species representing ca. 22% of vertebrate species observed in Denmark. Further, we demonstrate that storage conditions of the specimens accounted for ca. 50% of the total variation in mitochondrial DNA (mtDNA) preservation while the age of museum specimens had little effect: ca. 4%. In addition, we roughly estimate the cost of sequencing to be 25 EUR per specimen for obtaining sufficient amounts of DNA reads (ca. 200‐fold coverage) for reliable mitogenome assemblies while also obtaining low coverage genomic data. The large number of mitogenomes of Danish vertebrate species represents the initial groundwork for DNA‐based biodiversity assessments of vertebrates in Denmark and paves the way for practitioners to freely choose mitochondrial DNA markers.
Here, we present mitochondrial reference sequences for 182 Danish vertebrate species representing ca. 22% of vertebrates observed in Denmark. This is part of a larger DNA reference database, which aims to pave the way for future DNA‐based biodiversity assessments in Denmark.
Ancient and modern environmental DNA Pedersen, Mikkel Winther; Overballe-Petersen, Søren; Ermini, Luca ...
Philosophical transactions - Royal Society. Biological sciences,
01/2015, Volume:
370, Issue:
1660
Journal Article
Peer reviewed
Open access
DNA obtained from environmental samples such as sediments, ice or water (environmental DNA, eDNA), represents an important source of information on past and present biodiversity. It has revealed an ...ancient forest in Greenland, extended by several thousand years the survival dates for mainland woolly mammoth in Alaska, and pushed back the dates for spruce survival in Scandinavian ice-free refugia during the last glaciation. More recently, eDNA was used to uncover the past 50 000 years of vegetation history in the Arctic, revealing massive vegetation turnover at the Pleistocene/Holocene transition, with implications for the extinction of megafauna. Furthermore, eDNA can reflect the biodiversity of extant flora and fauna, both qualitatively and quantitatively, allowing detection of rare species. As such, trace studies of plant and vertebrate DNA in the environment have revolutionized our knowledge of biogeography. However, the approach remains marred by biases related to DNA behaviour in environmental settings, incomplete reference databases and false positive results due to contamination. We provide a review of the field.
Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of its targeted nature that allows ...sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample‐specific nucleotide identifiers are added to the amplicons prior to sequencing. The latter enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and during “library preparation”, that is, when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, ensuring that data generation is robust and fit for the chosen purpose is critically important for practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample‐specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms; one‐step PCR, two‐step PCR, and tagged PCR. Further, we distill the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications.
Infectious diseases cause significant production losses in aquaculture every year. Since the gut microbiota plays an essential role in regulating the host immune system, health and physiology, ...altered gut microbiota compositions are often associated with a diseased status. However, few studies have examined the association between disease severity and degree of gut dysbiosis, especially when the gut is not the site of the primary infection. Moreover, there is a lack of knowledge on whether bath treatment with formalin, a disinfectant commonly used in aquaculture to treat external infections, might affect the gut microbiome as a consequence of formalin ingestion. Here we investigate, through 16S rRNA gene metabarcoding, changes in the distal gut microbiota composition of a captive-reared cohort of 80 Atlantic salmon (Salmo salar L.), in consequence of an external bacterial skin infection due to a natural outbreak and subsequent formalin treatment.
We identified Tenacibaculum dicentrarchi as the causative disease pathogen and we show that the distal gut of diseased salmon presented a different composition from that of healthy individuals. A new, yet undescribed, Mycoplasma genus characterized the gut of healthy salmon, while in the sick fish we observed an increase in terms of relative abundance of Aliivibrio sp., a strain regarded as opportunistic. We also noticed a positive correlation between fish weight and Mycoplasma sp. relative abundance, potentially indicating a beneficial effect for its host. Moreover, we observed that the gut microbiota of fish treated with formalin was more similar to those of sick fish than healthy ones.
We conclude that external Tenacibaculum infections have the potential of indirectly affecting the host gut microbiota. As such, treatment optimization procedures should account for that. Formalin treatment is not an optimal solution from a holistic perspective, since we observe an altered gut microbiota in the treated fish. We suggest its coupling with a probiotic treatment aimed at re-establishing a healthy community. Lastly, we have observed a positive correlation of Mycoplasma sp. with salmon health and weight, therefore we encourage further investigations towards its potential utilization as a biomarker for monitoring health in salmon and potentially other farmed fish species.
Bacteria play an important role in the degradation of bone material. However, much remains to be learnt about the structure of their communities in degrading bone, and how the depositional ...environment influences their diversity throughout the exposure period. We genetically profiled the bacterial community in an experimental series of pig bone fragments (femur and humeri) deposited at different well-defined environments in Denmark. The bacterial community in the bone fragments and surrounding depositional environment were studied over one year, and correlated with the bioerosion damage patterns observed microscopically in the bones. We observed that the bacterial communities within the bones were heavily influenced by the local microbial community, and that the general bone microbial diversity increases with time after exposure. We found the presence of several known collagenase producing bacterial groups, and also observed increases in the relative abundance of several of these in bones with tunneling. We anticipate that future analyses using shotgun metagenomics on this and similar datasets will be able to provide insights into mechanisms of microbiome driven bone degradation.
The genomic origin of Zana of Abkhazia Margaryan, Ashot; Sinding, Mikkel‐Holger S.; Carøe, Christian ...
Advanced genetics,
June 2021, Volume:
2, Issue:
2
Journal Article
Peer reviewed
Open access
Enigmatic phenomena have sparked the imagination of people around the globe into creating folkloric creatures. One prime example is Zana of Abkhazia (South Caucasus), a well‐documented 19th century ...female who was captured living wild in the forest. Zana's appearance was sufficiently unusual, that she was referred to by locals as an Almasty—the analog of Bigfoot in the Caucasus. Although the exact location of Zana's burial site was unknown, the grave of her son, Khwit, was identified in 1971. The genomes of Khwit and the alleged Zana skeleton were sequenced to an average depth of ca. 3× using ancient DNA techniques. The identical mtDNA and parent‐offspring relationship between the two indicated that the unknown woman was indeed Zana. Population genomic analyses demonstrated that Zana's immediate genetic ancestry can likely be traced to present‐day East‐African populations. We speculate that Zana might have had a genetic disorder such as congenital generalized hypertrichosis which could partially explain her strange behavior, lack of speech, and long body hair. Our findings elucidate Zana's unfortunate story and provide a clear example of how prejudices of the time led to notions of cryptic hominids that are still held and transmitted by some today.
The displaced origins of a misconstrued person. Face reconstruction from Tkhina‐75 skull. Bottom right: artistic reconstruction after consideration of contemporary witness accounts.
Extant Canis lupus genetic diversity can be grouped into three phylogenetically distinct clades: Eurasian and American wolves and domestic dogs.1 Genetic studies have suggested these groups trace ...their origins to a wolf population that expanded during the last glacial maximum (LGM)1–3 and replaced local wolf populations.4 Moreover, ancient genomes from the Yana basin and the Taimyr peninsula provided evidence of at least one extinct wolf lineage that dwelled in Siberia during the Pleistocene.35 Previous studies have suggested that Pleistocene Siberian canids can be classified into two groups based on cranial morphology. Wolves in the first group are most similar to present-day populations, although those in the second group possess intermediate features between dogs and wolves.67 However, whether this morphological classification represents distinct genetic groups remains unknown. To investigate this question and the relationships between Pleistocene canids, present-day wolves, and dogs, we resequenced the genomes of four Pleistocene canids from Northeast Siberia dated between >50 and 14 ka old, including samples from the two morphological categories. We found these specimens cluster with the two previously sequenced Pleistocene wolves, which are genetically more similar to Eurasian wolves. Our results show that, though the four specimens represent extinct wolf lineages, they do not form a monophyletic group. Instead, each Pleistocene Siberian canid branched off the lineage that gave rise to present-day wolves and dogs. Finally, our results suggest the two previously described morphological groups could represent independent lineages similarly related to present-day wolves and dogs.
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•Pleistocene Siberian wolves represent multiple extinct evolutionary lineages•Pleistocene wolves share ancestry with arctic dogs and some East Asian wolves•A Paleolithic dog specimen is genetically similar to other Pleistocene wolves
Ramos-Madrigal et al. sequence the genomes of four Pleistocene Siberian wolves, two of which have divergent cranial morphologies. These canids represent multiple extinct lineages that dwelled in Siberia >50 ka ago and at least until 14.1 ka ago and that contributed to the genetic ancestry of arctic dogs and some East Asian wolves.
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