A quantitative protocol for DNA metabarcoding of springtails (Collembola)

Genome ◽  
2016 ◽  
Vol 59 (9) ◽  
pp. 705-723 ◽  
Author(s):  
Seikoh Saitoh ◽  
Hiroaki Aoyama ◽  
Saori Fujii ◽  
Haruki Sunagawa ◽  
Hideki Nagahama ◽  
...  
Keyword(s):  
Internal Control ◽  
Sequence Similarity ◽  
Community Sample ◽  
Pcr Primers ◽  
Dna Metabarcoding ◽  
Species Abundances ◽  
Collembolan Species ◽  
Order Degenerate ◽  
Number Of Individuals ◽  
Mock Communities

We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25–30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91–0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.

scholarly journals Switching Competitors Reduces Win-Stay but Not Lose-Shift Behaviour: The Role of Outcome-Action Association Strength on Reinforcement Learning

Games ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 25
Author(s):  
Vincent Srihaput ◽  
Kaylee Craplewe ◽  
Benjamin James Dyson
Keyword(s):  
Reinforcement Learning ◽  
Internal Control ◽  
Strong Association ◽  
Poor Quality ◽  
Group Data ◽  
Learning Principles ◽  
Zero Sum ◽  
Quality Decision ◽  
Number Of Individuals

Predictability is a hallmark of poor-quality decision-making during competition. One source of predictability is the strong association between current outcome and future action, as dictated by the reinforcement learning principles of win–stay and lose–shift. We tested the idea that predictability could be reduced during competition by weakening the associations between outcome and action. To do this, participants completed a competitive zero-sum game in which the opponent from the current trial was either replayed (opponent repeat) thereby strengthening the association, or, replaced (opponent change) by a different competitor thereby weakening the association. We observed that win–stay behavior was reduced during opponent change trials but lose–shiftbehavior remained reliably predictable. Consistent with the group data, the number of individuals who exhibited predictable behavior following wins decreased for opponent change relative to opponent repeat trials. Our data show that future actions are more under internal control following positive relative to negative outcomes, and that externally breaking the bonds between outcome and action via opponent association also allows us to become less prone to exploitation.

scholarly journals On the complexity of haplotyping a microbial community

2020 ◽  
Author(s):  
Samuel M Nicholls ◽  
Wayne Aubrey ◽  
Kurt De Grave ◽  
Leander Schietgat ◽  
Christopher J Creevey ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Community Sample ◽  
Population Level ◽  
Single Individual ◽  
Metagenomic Sample ◽  
Potential Benefits ◽  
Software Implementations ◽  
Occurrence Matrix ◽  
Number Of Individuals ◽  
Traversal Algorithm

Abstract Motivation Population-level genetic variation enables competitiveness and niche specialization in microbial communities. Despite the difficulty in culturing many microbes from an environment, we can still study these communities by isolating and sequencing DNA directly from an environment (metagenomics). Recovering the genomic sequences of all isoforms of a given gene across all organisms in a metagenomic sample would aid evolutionary and ecological insights into microbial ecosystems with potential benefits for medicine and biotechnology. A significant obstacle to this goal arises from the lack of a computationally tractable solution that can recover these sequences from sequenced read fragments. This poses a problem analogous to reconstructing the two sequences that make up the genome of a diploid organism (i.e. haplotypes), but for an unknown number of individuals and haplotypes. Results The problem of single individual haplotyping (SIH) was first formalised by Lancia et al. in 2001. Now, nearly two decades later, we discuss the complexity of “haplotyping” metagenomic samples, with a new formalisation of Lancia et al’s data structure that allows us to effectively extend the single individual haplotype problem to microbial communities. This work describes and formalizes the problem of recovering genes (and other genomic subsequences) from all individuals within a complex community sample, which we term the metagenomic individual haplotyping (MIH) problem. We also provide software implementations for a pairwise single nucleotide variant (SNV) co-occurrence matrix and greedy graph traversal algorithm. Availability and implementation Our reference implementation of the described pairwise SNV matrix (Hansel) and greedy haplotype path traversal algorithm (Gretel) are open source, MIT licensed and freely available online at github.com/samstudio8/hansel and github.com/samstudio8/gretel, respectively.

Species in the faeces: DNA metabarcoding as a method to determine the diet of the endangered yellow-eyed penguin

2020 ◽  
Vol 47 (6) ◽  
pp. 509
Author(s):  
Melanie J. Young ◽  
Ludovic Dutoit ◽  
Fiona Robertson ◽  
Yolanda van Heezik ◽  
Philip J. Seddon ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Diet Composition ◽  
Pcr Primers ◽  
Fish Prey ◽  
Genus Level ◽  
Faecal Samples ◽  
Significant Difference ◽  
Dna Metabarcoding ◽  
Reference Databases ◽  
Diet Assessment

Abstract Context. Diet variability is a significant driver of seabird decline; however, data on seabird diet composition and trends have been affected by changes in precision and resolution owing to the evolution of different sampling methods over time. We investigated the effectiveness of applying a passive molecular diet method using faeces obtained from the endangered yellow-eyed penguin. Aims. To assess the feasibility of applying DNA metabarcoding methods to yellow-eyed penguin faeces to evaluate diet, and to compare the reliability of diet results derived from adults and chicks, and from latrine versus fresh faecal samples. Methods. We collected 313 faecal samples from yellow-eyed penguins resident on the Otago coast of New Zealand from October 2016 to August 2017. We used polymerase chain reaction (PCR) with mitochondrial 16S cephalopod and chordate primers to amplify prey DNA present in the faecal samples, and tested the completeness of our assembled reference databases based on previous diet research. Amplified prey DNA sequences were then assigned to taxa from our reference databases by using QIIME2. Key results. Mitochondrial 16S chordate PCR primers were effective at identifying 29 fish taxa, with 98.3% of amplified sequences being identified to species or genus level in 193 samples (61.7% collected). There was no significant difference in the number, occurrence or proportion of ray-finned fish prey DNA sequences derived from fresh samples or latrines. Mitochondrial 16S cephalopod PCR primers classified 1.98% of amplified DNA sequences as targets, with 96.5% of these target sequences being identified to species or genus level in 48 samples (15.3% collected), and five taxa identified. Conclusions. We recommend the collection of latrine samples to enable long-term monitoring of the diet of yellow-eyed penguins, which will optimise the trade-off between wildlife disturbance and dietary resolution. Further refinement is needed to identify cephalopod dietary components for yellow-eyed penguins, because our cephalopod primers were not as specific as those used for ray-finned fishes, amplifying a large number (>98%) of non-cephalopod species. Implications. DNA metabarcoding offers a robust and comprehensive alternative to other, more intrusive, seabird diet-assessment methods, but still requires parallel studies to provide critical information on prey size, true diet composition and diet quality.

scholarly journals Switching competitors reduces win-stay but not lose-shift behaviour: The role of outcome-action association strength on reinforcement learning

2020 ◽  
Author(s):  
Vincent Srihaput ◽  
Kaylee Craplewe ◽  
Ben Dyson
Keyword(s):  
Reinforcement Learning ◽  
Internal Control ◽  
Strong Association ◽  
Poor Quality ◽  
Group Data ◽  
Learning Principles ◽  
Zero Sum ◽  
Quality Decision ◽  
Number Of Individuals

Predictability is a hallmark of poor-quality decision-making during competition. One source of predictability is the strong association between current outcome and future action, as dictated by the reinforcement learning principles of win-stay and lose-shift. We tested the idea that predictability could be reduced during competition by weakening the associations between outcome and action. To do this, participants completed a competitive zero-sum game in which the opponent from the current trial was either replayed (opponent repeat) thereby strengthening the association, or, replaced (opponent change) by a different competitor thereby weakening the association. We observed that win-stay behaviour was reduced during opponent change trials but lose-shift behaviour remained reliably predictable. Consistent with the group data, the number of individuals who exhibited predictable behaviour following wins decreased for opponent change relative to opponent repeat trials. Our data show that future actions are more under internal control following positive relative to negative outcomes, and that externally breaking the bonds between outcome and action via opponent association also allow us to become less prone to exploitation.

scholarly journals Hyaloperonospora camelinae on Camelina sativa in Washington State: Detection, Seed Transmission, and Chemical Control

Plant Disease ◽  
2012 ◽  
Vol 96 (11) ◽  
pp. 1670-1674 ◽  
Author(s):  
E. M. Babiker ◽  
S. H. Hulbert ◽  
T. C. Paulitz
Keyword(s):  
Sequence Similarity ◽  
Seed Transmission ◽  
Pcr Primers ◽  
Camelina Sativa ◽  
Washington State ◽  
Its2 Region ◽  
Disease Symptoms ◽  
Light Microscopic Observation ◽  
Causal Pathogen ◽  
Polymerase Chain

Camelina (Camelina sativa) plants with symptoms of downy mildew were obtained from three different locations in Washington State. Based on polymerase chain reaction (PCR) and sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, the causal pathogen was identified as Hyaloperonospora camelinae. The PCR primers consistently amplified 699-bp bands from the infected plants but not from the asymptomatic plants. A comparison of the sequences with those in GenBank revealed 100% sequence similarity to H. camelinae. Growth and development of the H. camelinae was observed in different tissues using light microscopy and scanning electron microscopy (SEM). Light microscopic observation revealed the presence of oospores in the infected leaves and SEM revealed the presence of conidia and conidiophores on the seed surface. To determine whether H. camelinae is a seed-transmitted pathogen, seed collected from infected plants were planted in Sunshine professional growing mix maintained in a growth chamber. Disease symptoms were observed in 96% of the seedlings compared with 3% of the seedlings grown from seed from asymptomatic plants, which indicates that H. camelinae is a seed-transmitted pathogen. Seed treated with mefenoxam, a fungicide specific for Oomycetes, significantly reduced the incidence of the disease.

scholarly journals A broad range of human papillomavirus types detected with a general PCR method suitable for analysis of cutaneous tumours and normal skin

1999 ◽  
Vol 80 (9) ◽  
pp. 2437-2443 ◽  
Author(s):  
Ola Forslund ◽  
Annika Antonsson ◽  
Peter Nordin ◽  
Bo Stenquist ◽  
Bengt Göran Hansson
Keyword(s):  
Sequence Similarity ◽  
Normal Skin ◽  
Pcr Primers ◽  
Skin Surface ◽  
Skin Lesions ◽  
Human Papillomaviruses ◽  
Reading Frame ◽  
Skin Biopsies ◽  
Pcr Method ◽  
Hpv Types

A pair of degenerate PCR primers (FAP59/64) was designed from two relatively conserved regions of the L1 open reading frame of most human papillomaviruses (HPV). The size of the generated amplicon was about 480 bp. PCR using these primers was found capable of amplifying DNA from 87% (65/75) of the HPV types tested, its sensitivity being 1–10 copies for HPV-5, -20 and -30 clones. HPV was found in 63% (5/8) of tumour samples and in 63% (5/8) of normal skin biopsies from patients with various cutaneous tumours. HPV-5, HPV-8, HPV-12, HPVvs20-4 and six putatively novel HPV types were identified. No correlation was found to exist between specific HPV and tumour types. Skin surface swab samples from one or more sites on three of four healthy volunteers were found to contain HPV, types 12 and 49 being identified, as well as eight novel HPV types, two of which were also found among the patients. In all, HPV was detected in 75% (9/12) of those tested, five HPV types and 12 novel candidate types being identified, and 37% (7/19) of HPV-positive samples were found to manifest more than one HPV type. All the HPV detected manifested high degrees of nucleotide sequence similarity with HPV types associated with skin lesions and epidermodysplasia verruciformis. The overall HPV finding in the skin samples was 50% (20/40) using the FAP primers as compared to 18% (7/40) using another PCR test designed for skin types. The results thus suggest the new method to be sensitive and generally applicable for detecting cutaneous HPV.

scholarly journals Random sampling causes the low reproducibility of rare eukaryotic OTUs in Illumina COI metabarcoding

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3006 ◽  
Author(s):  
Matthieu Leray ◽  
Nancy Knowlton
Keyword(s):  
Random Sampling ◽  
Marine Invertebrates ◽  
Illumina Miseq ◽  
Pcr Primers ◽  
Biodiversity Monitoring ◽  
Analysis Pipeline ◽  
Operational Taxonomic Units ◽  
Abundance Patterns ◽  
Dna Metabarcoding ◽  
Data Reproducibility

DNA metabarcoding, the PCR-based profiling of natural communities, is becoming the method of choice for biodiversity monitoring because it circumvents some of the limitations inherent to traditional ecological surveys. However, potential sources of bias that can affect the reproducibility of this method remain to be quantified. The interpretation of differences in patterns of sequence abundance and the ecological relevance of rare sequences remain particularly uncertain. Here we used one artificial mock community to explore the significance of abundance patterns and disentangle the effects of two potential biases on data reproducibility: indexed PCR primers and random sampling during Illumina MiSeq sequencing. We amplified a short fragment of the mitochondrial Cytochrome c Oxidase Subunit I (COI) for a single mock sample containing equimolar amounts of total genomic DNA from 34 marine invertebrates belonging to six phyla. We used seven indexed broad-range primers and sequenced the resulting library on two consecutive Illumina MiSeq runs. The total number of Operational Taxonomic Units (OTUs) was ∼4 times higher than expected based on the composition of the mock sample. Moreover, the total number of reads for the 34 components of the mock sample differed by up to three orders of magnitude. However, 79 out of 86 of the unexpected OTUs were represented by <10 sequences that did not appear consistently across replicates. Our data suggest that random sampling of rare OTUs (e.g., small associated fauna such as parasites) accounted for most of variation in OTU presence–absence, whereas biases associated with indexed PCRs accounted for a larger amount of variation in relative abundance patterns. These results suggest that random sampling during sequencing leads to the low reproducibility of rare OTUs. We suggest that the strategy for handling rare OTUs should depend on the objectives of the study. Systematic removal of rare OTUs may avoid inflating diversity based on commonβdescriptors but will exclude positive records of taxa that are functionally important. Our results further reinforce the need for technical replicates (parallel PCR and sequencing from the same sample) in metabarcoding experimental designs. Data reproducibility should be determined empirically as it will depend upon the sequencing depth, the type of sample, the sequence analysis pipeline, and the number of replicates. Moreover, estimating relative biomasses or abundances based on read counts remains elusive at the OTU level.

scholarly journals Replicate DNA metabarcoding can discriminate seasonal and spatial abundance shifts in river macroinvertebrate assemblages

2021 ◽  
Vol 4 ◽  
Author(s):  
Alex Bush ◽  
Zacchaeus Compson ◽  
Matilda Kattilakoski ◽  
Natalie Rideout ◽  
Brianna Levenstein ◽  
...  
Keyword(s):  
Predatory Fish ◽  
Great Promise ◽  
Macroinvertebrate Communities ◽  
Ecological Studies ◽  
Biodiversity Data ◽  
Occurrence Data ◽  
Dna Metabarcoding ◽  
Species Abundances ◽  
Fine Resolution ◽  
Small Tributary

Metabarcoding is capable of delivering consistent and accurate fine-resolution biodiversity data, and offers great promise for improving aspects of environmental assessment and research. Even so, many ecologists are keen to make further inferences about species’ abundances and the number of sequence reads has proven to be a poor proxy for abundance. The conservative interpretation has been to treat metabarcoding data as presence/absence, and although such data are less rich, occurrence and abundance are only different expressions of the same phenomenon. Interestingly if we assume the probability of detecting individuals is constant, it should be possible to use changes in the frequency of detection to infer changes in the underlying abundance. We tested the possibility that changes in the abundance structure of benthic macroinvertebrate communities could be recovered using replicated metabarcoding. We conducted 5 monthly surveys from Jun-Nov 2019 at the Catamaran Brook, a small tributary of the Little Southwest Miramichi River in New Brunswick, Canada. Each survey collected 30 benthic samples divided between control and treatment cages that excluded predatory fish. A further 6 samples were taken for traditional microscopic identification and counting. Analysis of the metabarcoding data demonstrated that we could recover plausible changes in abundance from occurrence data, including significant responses to both seasonal dynamics and the experimental exclusion of predators. The microscopy samples merely confirmed that count data are highly stochastic, and therefore while specific estimates of expected abundance from our model are highly uncertain, they capture those differences we could validate. In summary, while we confirmed that occurrence data are more robust for routine bioassessment, it is possible to recover fine-resolution changes in abundance that can inform ecological studies using metabarcoding.

scholarly journals Profiling grapevine trunk pathogens in planta: A case for community-targeted DNA metabarcoding

2018 ◽  
Author(s):  
Abraham Morales-Cruz ◽  
Rosa Figueroa-Balderas ◽  
Jadran F. García ◽  
Eric Tran ◽  
Philippe E. Rolshausen ◽  
...  
Keyword(s):  
Microbial Ecology ◽  
Time Course ◽  
Universal Primers ◽  
In Planta ◽  
Universal Primer ◽  
Grapevine Trunk Diseases ◽  
Field Samples ◽  
Dna Metabarcoding ◽  
Trunk Diseases ◽  
Mock Communities

ABSTRACTDNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Their detection is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the Fungal Kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated Ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology. We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were greatly over- or under-estimated with universal primers. Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.

Sign in / Sign up

Export Citation Format

Share Document