scholarly journals Evaluating intraspecific genetic diversity of a fish population using environmental DNA: An approach to distinguish true haplotypes from erroneous sequences

2018 ◽  
Author(s):  
Satsuki Tsuji ◽  
Masaki Miya ◽  
Masayuki Ushio ◽  
Hirotoshi Sato ◽  
Toshifumi Minamoto ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
False Positive ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
Detection Accuracy ◽  
Sequence Variant ◽  
Plecoglossus Altivelis ◽  
Detection Rates ◽  
Loop Region ◽  
Intraspecific Genetic Diversity

AbstractRecent advances in environmental DNA (eDNA) analysis using high-throughput sequencing (HTS) provide a non-invasive way to evaluate the intraspecific genetic diversity of aquatic macroorganisms. However, erroneous sequences present in HTS data can result in false positive haplotypes; therefore, reliable strategies are necessary to eliminate such erroneous sequences when evaluating intraspecific genetic diversity using eDNA metabarcoding. In this study, we propose an approach combining denoising using amplicon sequence variant (ASV) method and the removal of haplotypes with low detection rates. A mixture of rearing water of Ayu (Plecoglossus altivelis altivelis) was used as an eDNA sample. In total, nine haplotypes of Ayu mitochondrial D-loop region were contained in the sample and amplified by two-step tailed PCR. The 15 PCR replicates indexed different tags were prepared from the eDNA sample to compare the detection rates between true haplotypes and false positive haplotypes. All PCR replications were sequenced by HTS, and the total number of detected true haplotypes and false positive haplotypes were compared with and without denoising using the two types of ASV methods, Divisive Amplicon Denoising Algorithm 2 (DADA2) and UNOISE3. The use of both ASV methods considerably reduced the number of false positive haplotypes. Moreover, all true haplotypes were detected in all 15 PCR, whereas false positive haplotypes had detection rates varying from 1/15 to 15/15. Thus, by removing haplotypes with lower detection rates than 15/15, the number of false positive haplotypes were further reduced. The approach proposed in this study successfully eliminated most of false positive haplotypes in the HTS data obtained from eDNA samples, which allowed us to improve the detection accuracy for evaluating intraspecific genetic diversity using eDNA analysis.

scholarly journals Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

2019 ◽  
Author(s):  
Satsuki Tsuji ◽  
Atsushi Maruyama ◽  
Masaki Miya ◽  
Masayuki Ushio ◽  
Hirotoshi Sato ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Water Sample ◽  
Sanger Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Environmental Dna ◽  
Intraspecific Diversity ◽  
Survey Method ◽  
Intraspecific Genetic Diversity

AbstractEnvironmental DNA (eDNA) analysis has recently been used as a new tool for estimating intraspecific diversity. However, whether known haplotypes contained in a sample can be detected correctly using eDNA-based methods has been examined only by an aquarium experiment. Here, we tested whether the haplotypes of Ayu fish (Plecoglossus altivelis altivelis) detected in a capture survey could also be detected from an eDNA sample derived from the field that contained various haplotypes with low concentrations and foreign substances. A water sample and Ayu specimens collected from a river on the same day were analysed by eDNA analysis and Sanger sequencing, respectively. The 10 L water sample was divided into 20 filters for each of which 15 PCR replications were performed. After high-throughput sequencing, denoising was performed using two of the most widely used denoising packages, UNOISE3 and DADA2. Of the 42 haplotypes obtained from the Sanger sequencing of 96 specimens, 38 (UNOISE3) and 41 (DADA2) haplotypes were detected by eDNA analysis. When DADA2 was used, except for one haplotype, haplotypes owned by at least two specimens were detected from all the filter replications. This study showed that the eDNA analysis for evaluating intraspecific genetic diversity provides comparable results for large-scale capture-based conventional methods, suggesting that it could become a more efficient survey method for investigating intraspecific genetic diversity in the field.

scholarly journals Evaluating intraspecific genetic diversity using environmental DNA and denoising approach: A case study using tank water

2019 ◽  
Vol 2 (1) ◽  
pp. 42-52 ◽  
Author(s):  
Satsuki Tsuji ◽  
Masaki Miya ◽  
Masayuki Ushio ◽  
Hirotoshi Sato ◽  
Toshifumi Minamoto ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Environmental Dna ◽  
Intraspecific Genetic Diversity ◽  
Tank Water

scholarly journals Estimating intraspecific genetic diversity from community DNA metabarcoding data

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4644 ◽  
Author(s):  
Vasco Elbrecht ◽  
Ecaterina Edith Vamos ◽  
Dirk Steinke ◽  
Florian Leese
Keyword(s):  
Genetic Diversity ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Intraspecific Diversity ◽  
Great Promise ◽  
Mock Community ◽  
Dna Metabarcoding ◽  
Intraspecific Genetic Diversity ◽  
Primer Sets ◽  
Haplotype Information

BackgroundDNA metabarcoding is used to generate species composition data for entire communities. However, sequencing errors in high-throughput sequencing instruments are fairly common, usually requiring reads to be clustered into operational taxonomic units (OTUs), losing information on intraspecific diversity in the process. While Cytochrome c oxidase subunit I (COI) haplotype information is limited in resolving intraspecific diversity it is nevertheless often useful e.g. in a phylogeographic context, helping to formulate hypotheses on taxon distribution and dispersal.MethodsThis study combines sequence denoising strategies, normally applied in microbial research, with additional abundance-based filtering to extract haplotype information from freshwater macroinvertebrate metabarcoding datasets. This novel approach was added to the R package “JAMP” and can be applied to COI amplicon datasets. We tested our haplotyping method by sequencing (i) a single-species mock community composed of 31 individuals with 15 different haplotypes spanning three orders of magnitude in biomass and (ii) 18 monitoring samples each amplified with four different primer sets and two PCR replicates.ResultsWe detected all 15 haplotypes of the single specimens in the mock community with relaxed filtering and denoising settings. However, up to 480 additional unexpected haplotypes remained in both replicates. Rigorous filtering removes most unexpected haplotypes, but also can discard expected haplotypes mainly from the small specimens. In the monitoring samples, the different primer sets detected 177–200 OTUs, each containing an average of 2.40–3.30 haplotypes per OTU. The derived intraspecific diversity data showed population structures that were consistent between replicates and similar between primer pairs but resolution depended on the primer length. A closer look at abundant taxa in the dataset revealed various population genetic patterns, e.g. the stoneflyTaeniopteryx nebulosaand the caddisflyHydropsyche pellucidulashowed a distinct north–south cline with respect to haplotype distribution, while the beetleOulimnius tuberculatusand the isopodAsellus aquaticusdisplayed no clear population pattern but differed in genetic diversity.DiscussionWe developed a strategy to infer intraspecific genetic diversity from bulk invertebrate metabarcoding data. It needs to be stressed that at this point this metabarcoding-informed haplotyping is not capable of capturing the full diversity present in such samples, due to variation in specimen size, primer bias and loss of sequence variants with low abundance. Nevertheless, for a high number of species intraspecific diversity was recovered, identifying potentially isolated populations and taxa for further more detailed phylogeographic investigation. While we are currently lacking large-scale metabarcoding datasets to fully take advantage of our new approach, metabarcoding-informed haplotyping holds great promise for biomonitoring efforts that not only seek information about species diversity but also underlying genetic diversity.

scholarly journals Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

2020 ◽  
Vol 20 (5) ◽  
pp. 1248-1258 ◽  
Author(s):  
Satsuki Tsuji ◽  
Atsushi Maruyama ◽  
Masaki Miya ◽  
Masayuki Ushio ◽  
Hirotoshi Sato ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Dna Analysis ◽  
Environmental Dna ◽  
Fish Population ◽  
High Potential ◽  
Wild Fish ◽  
Intraspecific Genetic Diversity

Predicting Fetal Chromosome Anomalies in the First Trimester Using Pregnancy Associated Plasma Protein-A: A Comparison of Statistical Methods

1993 ◽  
Vol 32 (02) ◽  
pp. 175-179 ◽  
Author(s):  
B. Brambati ◽  
T. Chard ◽  
J. G. Grudzinskas ◽  
M. C. M. Macintosh
Keyword(s):  
Logistic Regression ◽  
General Population ◽  
Likelihood Ratio ◽  
False Positive ◽  
False Positive Rate ◽  
Ratio Method ◽  
Detection Rates ◽  
Gaussian Distributions ◽  
Positive Rate ◽  
Likelihood Ratio Method

Abstract:The analysis of the clinical efficiency of a biochemical parameter in the prediction of chromosome anomalies is described, using a database of 475 cases including 30 abnormalities. A comparison was made of two different approaches to the statistical analysis: the use of Gaussian frequency distributions and likelihood ratios, and logistic regression. Both methods computed that for a 5% false-positive rate approximately 60% of anomalies are detected on the basis of maternal age and serum PAPP-A. The logistic regression analysis is appropriate where the outcome variable (chromosome anomaly) is binary and the detection rates refer to the original data only. The likelihood ratio method is used to predict the outcome in the general population. The latter method depends on the data or some transformation of the data fitting a known frequency distribution (Gaussian in this case). The precision of the predicted detection rates is limited by the small sample of abnormals (30 cases). Varying the means and standard deviations (to the limits of their 95% confidence intervals) of the fitted log Gaussian distributions resulted in a detection rate varying between 42% and 79% for a 5% false-positive rate. Thus, although the likelihood ratio method is potentially the better method in determining the usefulness of a test in the general population, larger numbers of abnormal cases are required to stabilise the means and standard deviations of the fitted log Gaussian distributions.

scholarly journals Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Ryohei Nakao ◽  
Hideyuki Doi ◽  
Toshifumi Minamoto
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Correlation Coefficients ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Taqman Probe ◽  
Individual Species ◽  
Natural Environments ◽  
Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

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