{"title":"Genome and Metagenome Skimming: Future Sequencing Methods for Environmental DNA (eDNA) Studies.","authors":"Yiqiu Lu, Yuran Dong, Min Zhang, Lingfeng Mao","doi":"10.1111/1755-0998.14095","DOIUrl":"https://doi.org/10.1111/1755-0998.14095","url":null,"abstract":"<p><p>Genome skimming (GS), also referred to as low-coverage shotgun sequencing, is an efficient and cost-effective sequencing method that targets high-copy regions in genomes. It is most commonly used for species identification, phylogenetic analysis and expansion of reference libraries. GS can be applied to single species or composite DNA samples representing multiple species; the latter is termed metagenome skimming (MGS). GS/MGS shows promise as an effective approach for environmental DNA (eDNA) studies, but it is currently limited to ancient sedimentary samples. There is the potential to expand this methodology to other eDNA sources, including water, soil and airborne samples. In this paper, we introduce GS/MGS and briefly review its current applications. We also discuss the potential benefits and challenges of using GS/MGS to assay eDNA. eDNA GS/MGS is a promising technology that could broaden eDNA studies if some methodological challenges can be addressed.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e14095"},"PeriodicalIF":5.5,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143571621","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
K Birchard, C Boccia, H Lounder, L Colston-Nepali, V L Friesen
{"title":"Popfinder: A Highly Effective Artificial Neural Network Package for Genetic Population Assignment.","authors":"K Birchard, C Boccia, H Lounder, L Colston-Nepali, V L Friesen","doi":"10.1111/1755-0998.14096","DOIUrl":"https://doi.org/10.1111/1755-0998.14096","url":null,"abstract":"<p><p>The ability to assign biological samples to source populations with high accuracy and precision based on genetic variation is important for numerous applications from ecological studies through wildlife conservation to epidemiology. However, population assignment when genetic differentiation is low is challenging, and methods to address this problem are lacking. The application of artificial neural networks to population assignment using genomic data is highly promising. Here we present popfinder: a new, easy-to-use Python-based artificial neural network pipeline for genetic population assignment. We tested popfinder both with simulated genetic data from populations connected by varying levels of gene flow and with reduced-representation sequence data for three species of seabirds with weak to no population genetic structure. Popfinder was able to assign individuals to their source populations with high accuracy, precision and recall in most cases, including both simulated and empirical data sets, except in the empirical data set with the weakest population structure, where the comparator programs also performed poorly. Compared to other available software, popfinder was slower on the simulated data sets due to hyperparameter tuning and the fact that it does not reduce the dimensionality of the data set; however, all programs ran in seconds on empirical data sets. Additionally, popfinder provides a perturbation ranking method to help develop optimised SNP panels for genetic population assignment and is designed to be user-friendly. Finally, we caution users of all assignment programs to watch both for leakage of data during model training, which can lead to overfitting and inflation of performance metrics, and for unequal detection probabilities.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e14096"},"PeriodicalIF":5.5,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143571622","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cassandra Elphinstone, Rob Elphinstone, Marco Todesco, Loren H Rieseberg
{"title":"RepeatOBserver: Tandem Repeat Visualisation and Putative Centromere Detection.","authors":"Cassandra Elphinstone, Rob Elphinstone, Marco Todesco, Loren H Rieseberg","doi":"10.1111/1755-0998.14084","DOIUrl":"https://doi.org/10.1111/1755-0998.14084","url":null,"abstract":"<p><p>Tandem repeats play an important role in centromere structure, subtelomeric regions, DNA methylation, recombination and the regulation of gene activity. Analysis of their distribution in genomes offers a potential means for predicting putative centromere locations, which continues to be a challenge for genome annotation. Here we present RepeatOBserver (https://github.com/celphin/RepeatOBserverV1), a new tool for visualising repeat patterns and identifying putative centromere locations, using a Fourier transform of DNA walks. RepeatOBserver can identify and visualise a broad range of perfect and imperfect repeats (3-5000 bp long) in genome assemblies without any a priori knowledge of repeat sequences or the need for optimising parameters. RepeatOBserver heatmaps can distinguish between tandem and retrotransposon repeats. We analysed 159 chromosomes with experimentally-verified centromere positions from 12 plant and animal species. We find that 93% of experimentally-verified tandem repeat centromeres occur in regions of low sequence diversity and 97% of retrotransposon centromeres occur in regions with a high abundance of repeat lengths. Depending on the centromere type predicted by the heatmaps, putative centromere locations can be predicted using either a genomic Shannon diversity index or a repeat abundance sum. RepeatOBserver can also locate other regions of interest including potential neocentromeres and gene copy variation. Split and inverted tandem repeats at inversion boundaries suggest that chromosomal inversions or mis-assemblies can also be located. RepeatOBserver is a flexible tool for comprehensive characterisation of repeat patterns that can be used to visualise and identify a variety of regions of interest in genome assemblies.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e14084"},"PeriodicalIF":5.5,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539580","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Fiifi Agyabeng-Dadzie, Megan S Beaudry, Alex Deyanov, Haley Slanis, Minh Q Duong, Randi Turner, Asis Khan, Cesar A Arias, Jessica C Kissinger, Travis C Glenn, Rodrigo de Paula Baptista
{"title":"Evaluating the Benefits and Limits of Multiple Displacement Amplification With Whole-Genome Oxford Nanopore Sequencing.","authors":"Fiifi Agyabeng-Dadzie, Megan S Beaudry, Alex Deyanov, Haley Slanis, Minh Q Duong, Randi Turner, Asis Khan, Cesar A Arias, Jessica C Kissinger, Travis C Glenn, Rodrigo de Paula Baptista","doi":"10.1111/1755-0998.14094","DOIUrl":"10.1111/1755-0998.14094","url":null,"abstract":"<p><p>Multiple displacement amplification (MDA) outperforms conventional PCR in long fragment and whole-genome amplification, making it attractive to couple MDA with long-read sequencing of samples with limited quantities of DNA to obtain improved genome assemblies. Here, we explore the efficacy and limits of MDA for efficient low-cost genome sequence assembly using Oxford Nanopore Technologies (ONTs) rapid library preparations and minION sequencing. We successfully generated almost complete genome sequences for all organisms examined, including Gram-positive (Staphylococcus aureus, Enterococcus faecium) and Gram-negative (Escherichia coli) prokaryotes and one challenging eukaryotic pathogen (Cryptosporidium spp) representing a broad spectrum of critical infectious disease pathogens. High-quality data from those samples were generated starting with only 0.025 ng of total DNA. Controlled sheared DNA samples exhibited a distinct pattern of size increase after MDA, which may be associated with the amplification of long, low-abundance fragments present in the assay, as well as generating concatemeric sequences during amplification. To address concatemers, we developed a computational pipeline (CADECT: Concatemer Detection Tool) to identify and remove putative concatemeric sequences. This study highlights the efficacy of MDA in generating high-quality genome assemblies from limited amounts of input DNA. Also, the CADECT pipeline effectively mitigated the impact of concatemeric sequences, enabling the assembly of contiguous sequences even in cases where the input genomic DNA was degraded. These results have significant implications for the study of organisms that are challenging to culture in vitro, such as Cryptosporidium, and for expediting critical results in clinical settings with limited quantities of available genomic DNA.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e14094"},"PeriodicalIF":5.5,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143522364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jennifer M Standley, Jose Marcelino, Fahong Yu, James D Ellis
{"title":"A Meta-Omics Approach Using eDNA and eRNA for the Assessment of Biotic Communities Associated With Royal Jelly Produced by the Western Honey Bee (Apis mellifera L.).","authors":"Jennifer M Standley, Jose Marcelino, Fahong Yu, James D Ellis","doi":"10.1111/1755-0998.14090","DOIUrl":"https://doi.org/10.1111/1755-0998.14090","url":null,"abstract":"<p><p>Royal jelly (RJ) is a glandular secretion fed to developing honey bee larvae by adult worker bees. It is also a potential source of disease transmission in and between honey bee colonies. We endeavored to characterize the microbiome, virome, and other biota present in RJ via an integrated meta-omics approach. Using a magnetic beads-based extraction protocol, we identified eDNA and eRNA fragments from organisms of interest in RJ using high-throughput metagenomics (DNA-seq), metatranscriptomics (total RNA-seq), and parallel sequencing. This allowed us to enhance the detection of Operational Taxonomic Units (OTUs) undetectable by standard 'omics or amplicon protocols'. Using this integrated approach, we detected OTUs present in RJ from honey bee pests and pathogens, including Melissococcus plutonius, Paenibacillus larvae, Varroa destructor, V. jacobsoni, Aethina tumida, Galleria mellonella, Vairimorpha ceranae, Apis mellifera filamentous virus, Black queen cell virus, Acute bee paralysis virus, Sacbrood virus, Deformed wing virus, Israeli acute bee paralysis virus, Kashmir bee virus, and Slow bee paralysis virus, as well as multiple beneficial gut bacteria from the genera Lactobacillus, Actinobacteria, and Gluconobacter. The presence of DNA and RNA from these organisms does not conclusively indicate the presence of live organisms in the RJ, but it does suggest some exposure of the RJ to these organisms. The results present a comprehensive eDNA and eRNA microbial profile of RJ, demonstrating that our novel method is an effective and sensitive molecular tool for high-resolution metagenomic and metatranscriptomic profiling, and is of value for detection of pathogens of concern for the beekeeping industry.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e14090"},"PeriodicalIF":5.5,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143514280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Thilina S Nimalrathna, Huan Fan, Ahimsa Campos-Arceiz, Akihiro Nakamura
{"title":"Dung Beetle iDNA Provides an Effective Way to Detect Diverse Mycological Communities.","authors":"Thilina S Nimalrathna, Huan Fan, Ahimsa Campos-Arceiz, Akihiro Nakamura","doi":"10.1111/1755-0998.14091","DOIUrl":"https://doi.org/10.1111/1755-0998.14091","url":null,"abstract":"<p><p>Fungi play crucial ecological and economic roles, yet their diversity and distribution remain poorly known and challenging to assess. Using recent advances in invertebrate-derived DNA (iDNA) for biodiversity monitoring, we investigated the potential of dung beetle iDNA for fungal sampling and monitoring. We sampled two habitats (rainforest vs. rubber plantation) and seasons (dry vs. rainy) in tropical Xishuangbanna, southwest China. We extracted, amplified and identified 9259 unique fungi Amplicon Sequence Variants (ASVs) from the gut of three species of dung beetles (Paragymnopleurus sp., telecoprids; Onthophagus diabolicus, paracoprids; and Onthophagus cf. gracilipes, endocoprids). Fungal community composition was different across habitats and seasons, with the highest diversity found in the rainy season rainforest. Our results were consistent with previous eDNA-based studies based on soil samples in the detection of habitat differences (both approaches were able to detect low diversity of Basidiomycota in rubber plantations). However, our approach outperformed soil-based eDNA studies in being able to detect fungal occurrences associated with seasonal precipitation patterns. Our findings highlight the utility of dung beetle iDNA to uncover spatiotemporal dynamics of fungal communities across different habitats. The use of iDNA broadens fungal biodiversity research, strengthens fungal monitoring to assess anthropogenic impacts and presents opportunities to conserve fungal diversity.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e14091"},"PeriodicalIF":5.5,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143497735","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Bayesian StairwayPlot for Inferring Single Population Demographic Histories From Site Frequency Spectra.","authors":"Sebastian Höhna, Ana Catalán","doi":"10.1111/1755-0998.14087","DOIUrl":"https://doi.org/10.1111/1755-0998.14087","url":null,"abstract":"<p><p>The StairwayPlot approach provides an elegant, flexible and powerful method to estimate complex demographic histories of single populations from site frequency spectrum data. It uses expected coalescent times to compute the expected site frequency spectrum within a multinomial likelihood function. Population sizes are allowed to vary freely between coalescent events but are constant within each interval. Here, we implement the StairwayPlot approach in the Bayesian software package RevBayes. We use approaches developed for Bayesian Skyline Plots, which include independent and identically distributed (i.i.d.) population sizes, Gaussian Markov random fields and Horseshoe Markov random fields as prior distributions on population sizes. Furthermore, we implement a recently developed approach for computing the leave-one-out cross-validation probability for efficient model selection. We compare inference from our Bayesian implementation to the original Maximum Likelihood implementation, StairwayPlot2. Our results show that our Bayesian implementation in RevBayes performs comparable to StairwayPlot2 in terms of parameter accuracy, which is expected given that both use the same underlying likelihood function. From our set of prior models, the Gaussian Markov random field prior performed best for smoothly varying demographic histories, while the Horseshoe Markov random field performs best for abruptly changing demographic histories. We conclude the study by exploring several choices often faced in empirical studies, including the estimate of the total sequence length, the assumed mutation rate, as well as biases through mis-calling ancestral alleles. We show using our empirical example that as few as 10 diploid individuals are sufficient to infer complex demographic histories, but at least 500 k single nucleotide polymorphisms (SNPs) are required.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e14087"},"PeriodicalIF":5.5,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143514282","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Selecting Competent Reverse Transcription Strategies to Maximise Biodiversity Recovery With eRNA Metabarcoding.","authors":"Fuwen Wang, Wei Xiong, Xuena Huang, Aibin Zhan","doi":"10.1111/1755-0998.14092","DOIUrl":"https://doi.org/10.1111/1755-0998.14092","url":null,"abstract":"<p><p>Both environmental DNA (eDNA) and environmental RNA (eRNA) have been widely adopted for biodiversity assessment. While eDNA often persists longer in environments, eRNA offers a more current view of biological activities. In eRNA metabarcoding, extracted eRNA is reverse transcribed into complementary DNA (cDNA) for metabarcoding. However, the efficacy of various reverse transcription strategies has not been evaluated. Here we compared the biodiversity recovery efficiency of three strategies: random priming with hexamers, oligo(dT) priming and taxa-specific priming using Mifish-U for fish in both high- and low-biodiversity regions. Our results demonstrate that reverse transcription strategies significantly impact biodiversity recovery. Random priming consistently detected the highest number of taxa in both low- and high-biodiversity regions. In low-biodiversity areas, oligo(dT) performed comparably to random hexamers; however, in high-biodiversity regions, random hexamers outperformed oligo(dT), particularly in recovering rare taxa. While taxa-specific priming was comparable to the other strategies for high-abundance taxa, it was less effective for rare taxa, thus limiting its utility for comprehensive biodiversity assessment. These differences are largely due to the multiple binding sites for random hexamers compared to the fewer or absent sites with oligo(dT) and taxa-specific primers under high eRNA degradation. Combining random hexamers and oligo(dT) significantly improved taxa recovery, especially for low-abundance species, supporting its best practice in eukaryotes. For prokaryotes or genes lacking polyadenylation, random priming is favoured over taxa- or gene-specific priming. Collectively, these findings underscore the critical importance of selecting appropriate reverse transcription strategies in eRNA metabarcoding, with significant implications for effective biodiversity monitoring and conservation efforts.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e14092"},"PeriodicalIF":5.5,"publicationDate":"2025-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143476185","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yves Bawin, Beyene Zewdie, Biruk Ayalew, Isabel Roldán-Ruiz, Steven B Janssens, Ayco J M Tack, Sileshi Nemomissa, Kassahun Tesfaye, Kristoffer Hylander, Olivier Honnay, Tom Ruttink
{"title":"A Molecular Survey of the Occurrence of Coffee Berry Disease Resistant Coffee Cultivars Near the Wild Gene Pool of Arabica Coffee in Its Region of Origin in Southwest Ethiopia.","authors":"Yves Bawin, Beyene Zewdie, Biruk Ayalew, Isabel Roldán-Ruiz, Steven B Janssens, Ayco J M Tack, Sileshi Nemomissa, Kassahun Tesfaye, Kristoffer Hylander, Olivier Honnay, Tom Ruttink","doi":"10.1111/1755-0998.14085","DOIUrl":"https://doi.org/10.1111/1755-0998.14085","url":null,"abstract":"<p><p>Cultivation of crops close to their wild relatives may jeopardise the integrity of wild genetic resources. Detecting cultivars among wild plants is necessary to characterise crop-wild gene flow, but can be challenging if cultivars and wild plants are phenotypically highly similar. Genomics tools can be used instead, but the selection of diagnostic loci for cultivar identification can be difficult if the wild and cultivated genepools are closely related. In Ethiopia, Arabica coffee cultivars resistant to coffee berry disease (CBD) occur near wild Coffea arabica plants and local landraces. However, the abundance and distribution of these cultivars across coffee sites remains unclear. Here, we present a new module of the SMAP package called SMAP relatedness pairwise to characterise pairwise genetic relationships between individuals based on haplotype calls and to identify diagnostic loci that distinguish (sets of) individuals from each other. Next, we estimate the relative abundance of CBD-resistant cultivars across 60 Ethiopian Arabica coffee sites using a genome-wide fingerprinting approach. We confirm the presence of these cultivars in around 75% of the coffee sites with a high agreement between a field survey and our DNA fingerprinting approach. At least 20 out of 60 sites with supposedly wild C. arabica individuals contain signatures of the cultivated genepool. Overall, we conclude that CBD-resistant cultivars are widespread in Ethiopian coffee sites. The development of SMAP relatedness pairwise opens opportunities to assess the distribution of coffee cultivars in other regions in Ethiopia and to apply similar screenings near wild relatives from other crops.</p>","PeriodicalId":211,"journal":{"name":"Molecular Ecology Resources","volume":" ","pages":"e14085"},"PeriodicalIF":5.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143466406","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}