共查询到20条相似文献,搜索用时 15 毫秒
1.
Wendy Wang Wei X. Tan Denis Bertrand Amanda H. Q. Ng Esther J. H. Boey Jayce J. Y. Koh Niranjan Nagarajan Rudolf Meier 《Molecular ecology resources》2018,18(5):1035-1049
DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well‐equipped molecular laboratory and is time‐consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION? and demonstrating that one flow cell can generate barcodes for ~500 specimens despite the high basecall error rates of MinION? reads. The pipeline overcomes these errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. Consensus barcodes are overall mismatch‐free but retain indel errors that are concentrated in homopolymeric regions. They are addressed with an optional error correction pipeline that is based on conserved amino acid motifs from publicly available barcodes. The effectiveness of this pipeline is documented by analysing reads from three MinION? runs that represent three different stages of MinION? development. They generated data for (i) 511 specimens of a mixed Diptera sample, (ii) 575 specimens of ants and (iii) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION? barcodes for 490 of the 511 specimens which were assessed against reference Sanger barcodes (N = 471). Overall, the MinION? barcodes have an accuracy of 99.3%–100% with the number of ambiguous bases after correction ranging from <0.01% to 1.5% depending on which correction pipeline is used. We demonstrate that it requires ~2 hr of sequencing to gather all information needed for obtaining reliable barcodes for most specimens (>90%). We estimate that up to 1,000 barcodes can be generated in one flow cell and that the cost per barcode can be 相似文献
2.
Sean W. J. Prosser Jeremy R. deWaard Scott E. Miller Paul D. N. Hebert 《Molecular ecology resources》2016,16(2):487-497
Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next‐generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century‐old type specimens of Lepidoptera by attempting to recover 164‐bp and 94‐bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories – high (164‐bp sequence), medium (94‐bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR‐based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens. 相似文献
3.
Kristine Bohmann Siavash Mirarab Vineet Bafna M. Thomas P. Gilbert 《Molecular ecology》2020,29(14):2521-2534
Genetic tools are increasingly used to identify and discriminate between species. One key transition in this process was the recognition of the potential of the ca 658bp fragment of the organelle cytochrome c oxidase I (COI) as a barcode region, which revolutionized animal bioidentification and lead, among others, to the instigation of the Barcode of Life Database (BOLD), containing currently barcodes from >7.9 million specimens. Following this discovery, suggestions for other organellar regions and markers, and the primers with which to amplify them, have been continuously proposed. Most recently, the field has taken the leap from PCR‐based generation of DNA references into shotgun sequencing‐based “genome skimming” alternatives, with the ultimate goal of assembling organellar reference genomes. Unfortunately, in genome skimming approaches, much of the nuclear genome (as much as 99% of the sequence data) is discarded, which is not only wasteful, but can also limit the power of discrimination at, or below, the species level. Here, we advocate that the full shotgun sequence data can be used to assign an identity (that we term for convenience its “DNA‐mark”) for both voucher and query samples, without requiring any computationally intensive pretreatment (e.g. assembly) of reads. We argue that if reference databases are populated with such “DNA‐marks,” it will enable future DNA‐based taxonomic identification to complement, or even replace PCR of barcodes with genome skimming, and we discuss how such methodology ultimately could enable identification to population, or even individual, level. 相似文献
4.
Chao Xu Wenpan Dong Shuo Shi Tao Cheng Changhao Li Yanlei Liu Ping Wu Hongkun Wu Peng Gao Shiliang Zhou 《Molecular ecology resources》2015,15(6):1366-1374
A well‐covered reference library is crucial for successful identification of species by DNA barcoding. The biggest difficulty in building such a reference library is the lack of materials of organisms. Herbarium collections are potentially an enormous resource of materials. In this study, we demonstrate that it is likely to build such reference libraries using the reconstructed (self‐primed PCR amplified) DNA from the herbarium specimens. We used 179 rosaceous specimens to test the effects of DNA reconstruction, 420 randomly sampled specimens to estimate the usable percentage and another 223 specimens of true cherries (Cerasus, Rosaceae) to test the coverage of usable specimens to the species. The barcode rbcLb (the central four‐sevenths of rbcL gene) and matK was each amplified in two halves and sequenced on Roche GS 454 FLX+. DNA from the herbarium specimens was typically shorter than 300 bp. DNA reconstruction enabled amplification fragments of 400–500 bp without bringing or inducing any sequence errors. About one‐third of specimens in the national herbarium of China (PE) were proven usable after DNA reconstruction. The specimens in PE cover all Chinese true cherry species and 91.5% of vascular species listed in Flora of China. It is very possible to build well‐covered reference libraries for DNA barcoding of vascular species in China. As exemplified in this study, DNA reconstruction and DNA‐labelled next‐generation sequencing can accelerate the construction of local reference libraries. By putting the local reference libraries together, a global library for DNA barcoding becomes closer to reality. 相似文献
5.
Rodrigo A Bertels F Heled J Noder R Shearman H Tsai P 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2008,363(1512):3893-3902
This new century's biology promises more of everything--more genes, more organisms, more species and, in short, more data. The flood of data challenges us to find better and quicker ways to summarize and analyse. Here, we present preliminary results and proofs of concept from three of our research projects that are motivated by our search for solutions to the perils of plenty. First, we discuss how models of evolution can accommodate change to better reflect the dynamics of sequence diversity, particularly when it is becoming a lot easier to obtain sequences at different times and across intervals where the probability of new mutations contributing to this diversity is high. Second, we describe our work on the use of a single locus for species delimitation; this research targets the new DNA-barcoding approach that aims to catalogue the entirety of life. We have developed a single-locus test based on the coalescent that tests the null hypothesis of panmixis. Finally, we discuss new sequencing technologies, the types of data available and the efficacy of alignment-free methods to estimate pairwise distances for phylogenetic analyses. 相似文献
6.
Ken Kraaijeveld Letty A. de Weger Marina Ventayol García Henk Buermans Jeroen Frank Pieter S. Hiemstra Johan T. den Dunnen 《Molecular ecology resources》2015,15(1):8-16
Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen‐allergic patients. Current pollen monitoring methods are microscope‐based, labour intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore, a more efficient, cost‐effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen, and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next‐generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring. 相似文献
7.
A goal of many environmental DNA barcoding studies is to infer quantitative information about relative abundances of different taxa based on sequence read proportions generated by high‐throughput sequencing. However, potential biases associated with this approach are only beginning to be examined. We sequenced DNA amplified from faeces (scats) of captive harbour seals (Phoca vitulina) to investigate whether sequence counts could be used to quantify the seals’ diet. Seals were fed fish in fixed proportions, a chordate‐specific mitochondrial 16S marker was amplified from scat DNA and amplicons sequenced using an Ion Torrent PGM?. For a given set of bioinformatic parameters, there was generally low variability between scat samples in proportions of prey species sequences recovered. However, proportions varied substantially depending on sequencing direction, level of quality filtering (due to differences in sequence quality between species) and minimum read length considered. Short primer tags used to identify individual samples also influenced species proportions. In addition, there were complex interactions between factors; for example, the effect of quality filtering was influenced by the primer tag and sequencing direction. Resequencing of a subset of samples revealed some, but not all, biases were consistent between runs. Less stringent data filtering (based on quality scores or read length) generally produced more consistent proportional data, but overall proportions of sequences were very different than dietary mass proportions, indicating additional technical or biological biases are present. Our findings highlight that quantitative interpretations of sequence proportions generated via high‐throughput sequencing will require careful experimental design and thoughtful data analysis. 相似文献
8.
Porazinska DL Giblin-Davis RM Esquivel A Powers TO Sung W Thomas WK 《Molecular ecology》2010,19(24):5521-5530
The general patterns of increasing biodiversity from the poles to the equator have been well documented for large terrestrial organisms such as plants and vertebrates but are largely unknown for microbiota. In contrast to macrobiota, microbiota have long been assumed to exhibit cosmopolitan, random distributions and a lack of spatial patterns. To evaluate the assumption, we conducted a survey of nematode diversity within the soil, litter and canopy habitats of the humid lowland tropical rainforest of Costa Rica using an ultrasequencing ecometagenetic approach at a species-equivalent taxonomic level. Our data indicate that both richness and diversity of nematode communities in the tropical rainforests of Costa Rica are high and exceed observed values from temperate ecosystems. The majority of nematode species were unknown to science, providing evidence for the presence of highly endemic (not cosmopolitan) species of still completely undiscovered biodiversity. Most importantly, the greater taxonomic resolution used here allowed us to reveal predictable habitat associations for specific taxa and thus gain insights into their nonrandom distribution patterns. 相似文献
9.
J. Daligault E. Stoetzel E. A. Bennett N. M.‐L. Côté V. Nicolas A. Lalis C. Denys E.‐M. Geigl T. Grange 《Molecular ecology resources》2017,17(3):405-417
We present a cost‐effective metabarcoding approach, aMPlex Torrent, which relies on an improved multiplex PCR adapted to highly degraded DNA, combining barcoding and next‐generation sequencing to simultaneously analyse many heterogeneous samples. We demonstrate the strength of these improvements by generating a phylochronology through the genotyping of ancient rodent remains from a Moroccan cave whose stratigraphy covers the last 120 000 years. Rodents are important for epidemiology, agronomy and ecological investigations and can act as bioindicators for human‐ and/or climate‐induced environmental changes. Efficient and reliable genotyping of ancient rodent remains has the potential to deliver valuable phylogenetic and paleoecological information. The analysis of multiple ancient skeletal remains of very small size with poor DNA preservation, however, requires a sensitive high‐throughput method to generate sufficient data. We show this approach to be particularly adapted at accessing this otherwise difficult taxonomic and genetic resource. As a highly scalable, lower cost and less labour‐intensive alternative to targeted sequence capture approaches, we propose the aMPlex Torrent strategy to be a useful tool for the genetic analysis of multiple degraded samples in studies involving ecology, archaeology, conservation and evolutionary biology. 相似文献
10.
Markus Ruhsam Hardeep S. Rai Sarah Mathews T. Gregory Ross Sean W. Graham Linda A. Raubeson Wenbin Mei Philip I. Thomas Martin F. Gardner Richard A. Ennos Peter M. Hollingsworth 《Molecular ecology resources》2015,15(5):1067-1078
Obtaining accurate phylogenies and effective species discrimination using a small standardized set of plastid genes is challenging in evolutionarily young lineages. Complete plastid genome sequencing offers an increasingly easy‐to‐access source of characters that helps address this. The usefulness of this approach, however, depends on the extent to which plastid haplotypes track morphological species boundaries. We have tested the power of complete plastid genomes to discriminate among multiple accessions of 11 of 13 New Caledonian Araucaria species, an evolutionarily young lineage where the standard DNA barcoding approach has so far failed and phylogenetic relationships have remained elusive. Additionally, 11 nuclear gene regions were Sanger sequenced for all accessions to ascertain the success of species discrimination using a moderate number of nuclear genes. Overall, fewer than half of the New Caledonian Araucaria species with multiple accessions were monophyletic in the plastid or nuclear trees. However, the plastid data retrieved a phylogeny with a higher resolution compared to any previously published tree of this clade and supported the monophyly of about twice as many species and nodes compared to the nuclear data set. Modest gains in discrimination thus are possible, but using complete plastid genomes or a small number of nuclear genes in DNA barcoding may not substantially raise species discriminatory power in many evolutionarily young lineages. The big challenge therefore remains to develop techniques that allow routine access to large numbers of nuclear markers scaleable to thousands of individuals from phylogenetically disparate sample sets. 相似文献
11.
Kristin Hardge Stefan Neuhaus Estelle S. Kilias Christian Wolf Katja Metfies Stephan Frickenhaus 《Molecular ecology resources》2018,18(2):204-216
Next‐generation sequencing is a common method for analysing microbial community diversity and composition. Configuring an appropriate sequence processing strategy within the variety of tools and methods is a nontrivial task and can considerably influence the resulting community characteristics. We analysed the V4 region of 18S rRNA gene sequences of marine samples by 454‐pyrosequencing. Along this process, we generated several data sets with QIIME, mothur, and a custom‐made pipeline based on DNAStar and the phylogenetic tree‐based PhyloAssigner. For all processing strategies, default parameter settings and punctual variations were used. Our results revealed strong differences in total number of operational taxonomic units (OTUs), indicating that sequence preprocessing and clustering had a major impact on protist diversity estimates. However, diversity estimates of the abundant biosphere (abundance of ≥1%) were reproducible for all conducted processing pipeline versions. A qualitative comparison of diatom genera emphasized strong differences between the pipelines in which phylogenetic placement of sequences came closest to light microscopy‐based diatom identification. We conclude that diversity studies using different sequence processing strategies are comparable if the focus is on higher taxonomic levels, and if abundance thresholds are used to filter out OTUs of the rare biosphere. 相似文献
12.
Tom Oosting Elena Hilario Maren Wellenreuther Peter A. Ritchie 《Ecology and evolution》2020,10(16):8643-8651
The more demanding requirements of DNA preservation for genomic research can be difficult to meet when field conditions limit the methodological approaches that can be used or cause samples to be stored in suboptimal conditions. Such limitations may increase rates of DNA degradation, potentially rendering samples unusable for applications such as genome‐wide sequencing. Nonetheless, little is known about the impact of suboptimal sampling conditions. We evaluated the performance of two widely used preservation solutions (1. DESS: 20% DMSO, 0.25 M EDTA, NaCl saturated solution, and 2. Ethanol >99.5%) under a range of storage conditions over a three‐month period (sampling at 1 day, 1 week, 2 weeks, 1 month, and 3 months) to provide practical guidelines for DNA preservation. DNA degradation was quantified as the reduction in average DNA fragment size over time (DNA fragmentation) because the size distribution of DNA segments plays a key role in generating genomic datasets. Tissues were collected from a marine teleost species, the Australasian snapper, Chrysophrys auratus. We found that the storage solution has a strong effect on DNA preservation. In DESS, DNA was only moderately degraded after three months of storage while DNA stored in ethanol showed high levels of DNA degradation already within 24 hr, making samples unsuitable for next‐generation sequencing. Here, we conclude that DESS was the most promising solution when storing samples for genomic applications. We recognize that the best preservation protocol is highly dependent on the organism, tissue type, and study design. We highly recommend performing similar experiments before beginning a study. This study highlights the importance of testing sample preservation protocols and provides both practical and economical advice to improve DNA preservation when sampling for genome‐wide applications. 相似文献
13.
Brent C. Emerson Francesco Cicconardi Pietro P. Fanciulli Peter J. A. Shaw 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2011,366(1576):2391-2402
There has been much recent interest and progress in the characterization of community structure and community assembly processes through the application of phylogenetic methods. To date most focus has been on groups of taxa for which some relevant detail of their ecology is known, for which community composition is reasonably easily quantified and where the temporal scale is such that speciation is not likely to feature. Here, we explore how we might apply a molecular genetic approach to investigate community structure and assembly at broad taxonomic and geographical scales, where we have little knowledge of species ecology, where community composition is not easily quantified, and where speciation is likely to be of some importance. We explore these ideas using the class Collembola as a focal group. Gathering molecular evidence for cryptic diversity suggests that the ubiquity of many species of Collembola across the landscape may belie greater community complexity than would otherwise be assumed. However, this morphologically cryptic species-level diversity poses a challenge for attempts to characterize diversity both within and among local species assemblages. Recent developments in high throughput parallel sequencing technology, combined with mtDNA barcoding, provide an advance that can bring together the fields of phylogenetic and phylogeographic analysis to bear on this problem. Such an approach could be standardized for analyses at any geographical scale for a range of taxonomic groups to quantify the formation and composition of species assemblages. 相似文献
14.
Katherine D. Balasingham Ryan P. Walter Nicholas E. Mandrak Daniel D. Heath 《Molecular ecology》2018,27(1):112-127
The extraction and characterization of DNA from aquatic environmental samples offers an alternative, noninvasive approach for the detection of rare species. Environmental DNA, coupled with PCR and next‐generation sequencing (“metabarcoding”), has proven to be very sensitive for the detection of rare aquatic species. Our study used a custom‐designed group‐specific primer set and next‐generation sequencing for the detection of three species at risk (Eastern Sand Darter, Ammocrypta pellucida; Northern Madtom, Noturus stigmosus; and Silver Shiner, Notropis photogenis), one invasive species (Round Goby, Neogobius melanostomus) and an additional 78 native species from two large Great Lakes tributary rivers in southern Ontario, Canada: the Grand River and the Sydenham River. Of 82 fish species detected in both rivers using capture‐based and eDNA methods, our eDNA method detected 86.2% and 72.0% of the fish species in the Grand River and the Sydenham River, respectively, which included our four target species. Our analyses also identified significant positive and negative species co‐occurrence patterns between our target species and other identified species. Our results demonstrate that eDNA metabarcoding that targets the fish community as well as individual species of interest provides a better understanding of factors affecting the target species spatial distribution in an ecosystem than possible with only target species data. Additionally, eDNA is easily implemented as an initial survey tool, or alongside capture‐based methods, for improved mapping of species distribution patterns. 相似文献
15.
Bruce E. Deagle Laurence J. Clarke John A. Kitchener Andrea M. Polanowski Andrew T. Davidson 《Molecular ecology resources》2018,18(3):391-406
DNA metabarcoding is an efficient method for measuring biodiversity, but the process of initiating long‐term DNA‐based monitoring programmes, or integrating with conventional programs, is only starting. In marine ecosystems, plankton surveys using the continuous plankton recorder (CPR) have characterized biodiversity along transects covering millions of kilometres with time‐series spanning decades. We investigated the potential for use of metabarcoding in CPR surveys. Samples (n = 53) were collected in two Southern Ocean transects and metazoans identified using standard microscopic methods and by high‐throughput sequencing of a cytochrome c oxidase subunit I marker. DNA increased the number of metazoan species identified and provided high‐resolution taxonomy of groups problematic in conventional surveys (e.g., larval echinoderms and hydrozoans). Metabarcoding also generally produced more detections than microscopy, but this sensitivity may make cross‐contamination during sampling a problem. In some samples, the prevalence of DNA from large plankton such as krill masked the presence of smaller species. We investigated adding a fixed amount of exogenous DNA to samples as an internal control to allow determination of relative plankton biomass. Overall, the metabarcoding data represent a substantial shift in perspective, making direct integration into current long‐term time‐series challenging. We discuss a number of hurdles that exist for progressing DNA metabarcoding from the current snapshot studies to the requirements of a long‐term monitoring programme. Given the power and continually increasing efficiency of metabarcoding, it is almost certain this approach will play an important role in future plankton monitoring. 相似文献
16.
17.
Identification of fern gametophytes is generally hampered by low morphological complexity. Here we explore an alternative: DNA‐based identification. We obtained a plastid rbcL sequence from a sterile gametophyte of unknown origin (cultivated for more than 30 years) and employed blast to determine its affinities. Using this approach, we identified the gametophyte as Osmunda regalis. To evaluate the robustness of this determination, and the usefulness of rbcL in differentiating among species, we conducted a phylogenetic analysis of osmundaceous fern sequences. Based on our results, it is evident that DNA‐based identification has considerable potential in exploring the ecology of fern gametophytes. 相似文献
18.
Fabian Pérez‐Miranda Omar Mejía Eduardo Soto‐Galera Héctor Espinosa‐Pérez Lubomír Piálek Oldřich Říčan 《Journal of Zoological Systematics and Evolutionary Research》2018,56(2):223-247
We provide a review of the systematics of Herichthys by evaluating the usefulness of several mitochondrial and nuclear genetic markers together with morphological data. The nDNA next‐generation sequencing ddRAD analysis together with the mtDNA cytochrome b gene provided well‐resolved and well‐supported phylogenies of Herichthys. On the other hand, the nDNA S7 introns have limited resolution and support and the COI barcoding analysis completely failed to recover all but one species of Herichthys as monophyletic. The COI barcoding as currently implemented is thus insufficient to distinguish clearly distinct species in the genus Herichthys that are supported by other molecular markers and by morphological characters. Based on our results, Herichthys is composed of 11 species and includes two main clades (the H. labridens and H. cyanoguttatus species groups). Herichthys bartoni is in many respects the most plesiomorphic species in the genus and has a conflicting phylogenetic position between mtDNA and nDNA markers, where the robust nDNA ddRAD data place it as a rather distant basal member of the H. labridens species group. The mtDNA of H. bartoni is on the other hand only slightly divergent from the sympatric and syntopic H. labridens, and the species thus probably have hybridized in the relatively recent past. The sympatric and syntopic Herichthys steindachneri and H. pame are supported as sister species. The Herichthys cyanoguttatus species group shows two well‐separated basal species (the northernmost H. minckleyi and the southernmost H. deppii) followed by the closely related and centrally distributed species H. cyanoguttatus, H. tepehua, H. carpintis, and H. tamasopoensis whose relationships differ between analyses and show likely hybridizations between themselves and the two basal species as suggested by conflicts between DNA analyses. Several instances of introgressions/hybridizations have also been found between the two main clades of Herichthys. 相似文献
19.
An emergent science on the brink of irrelevance: a review of the past 8 years of DNA barcoding 总被引:2,自引:0,他引:2
DNA barcoding has become a well-funded, global enterprise since its proposition as a technique for species identification, delimitation and discovery in 2003. However, the rapid development of next generation sequencing (NGS) has the potential to render DNA barcoding irrelevant because of the speed with which it generates large volumes of genomic data. To avoid obsolescence, the DNA barcoding movement must adapt to use this new technology. This review examines the DNA barcoding enterprise, its continued resistance to improvement and the implications of this on the future of the discipline. We present the consistent failure of DNA barcoding to recognize its limitations and evolve its methodologies, reducing the usefulness of the data produced by the movement and throwing into doubt its ability to embrace NGS. 相似文献
20.
DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research, in revealing both the structural and functional characteristics of genomes. In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics, systems biology and pharmacogenomics. The next-generation DNA sequencing method was first introduced by the 454 Company in 2003, immediately followed by the establishment of the Solexa and Solid techniques by other biotech companies. Though it has not been long since the first emergence of this technology, with the fast and impressive improvement, the application of this technology has extended to almost all fields of genomics research, as a rival challenging the existing DNA microarray technology. This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research. Supported by the National High-Tech Research Program of China (Grant No.2006AA020704) and Shanghai Science and Technology Commission (Grant No. 05DZ22201) 相似文献