Molecular Ecology Resources最新文献

Assessing and monitoring genetic diversity is vital for understanding the ecology and evolution of natural populations but is often challenging in animal and plant species due to technically and physically demanding tissue sampling. Although environmental DNA (eDNA) metabarcoding is a promising alternative to the traditional population genetic monitoring based on biological samples, its practical application remains challenging due to spurious sequences present in the amplicon data, even after data processing with the existing sequence filtering and denoising (error correction) methods. Here we developed a novel amplicon filtering approach that can effectively eliminate such spurious amplicon sequence variants (ASVs) in eDNA metabarcoding data. A simple simulation of eDNA metabarcoding processes was performed to understand the patterns of read count (abundance) distributions of true ASVs and their polymerase chain reaction (PCR)-generated artefacts (i.e., false-positive ASVs). Based on the simulation results, the approach was developed to estimate the abundance distributions of true and false-positive ASVs using Gaussian mixture models and to determine a statistically based threshold between them. The developed approach was implemented as an R package, gmmDenoise and evaluated using single-species metabarcoding datasets in which all or some true ASVs (i.e., haplotypes) were known. Example analyses using community (multi-species) metabarcoding datasets were also performed to demonstrate how gmmDenoise can be used to derive reliable intraspecific diversity estimates and population genetic inferences from noisy amplicon sequencing data. The gmmDenoise package is freely available in the GitHub repository (https://github.com/YSKoseki/gmmDenoise).

Next-generation sequencing (NGS) has the potential to transform mosquito-borne disease surveillance but remains under-utilised. This study introduces a comprehensive multi-loci metabarcoding-based MX (molecular xenomonitoring) approach to mosquito and arbovirus surveillance, enabling parallel identification of mosquito vectors, circulating arboviruses, and vertebrate hosts from bulk mosquito collections. The feasibility of this approach was demonstrated through its application to a large set (n = 110) of bulk field collections. This set was complemented by a number (n = 28) of single-species mosquito pools that had previously been screened for viruses using quantitative reverse transcription PCR (RT-qPCR) and metatranscriptomics. Universal alphavirus and flavivirus primer sets were used to screen for arboviruses in the resulting metabarcoding library. Viral amplicons were then indexed and combined with mosquito-specific (ITS2), universal invertebrate (COI), and vertebrate (Cyt b) barcode amplicons prior to sequencing. This approach confirmed the presence of all previously identified mosquito species, as well as those commonly misidentified morphologically, and enabled a degree of quantification regarding their relative physical abundance in each collection. Additionally, the developed approach identified a diverse vertebrate host community (18 species), demonstrating its potential for defining host preferences and, in tandem with the viral screens and associated vector data, understanding disease transmission pathways. Importantly, metabarcoding detected a diversity of regionally prevalent arboviruses and insect-specific viruses, with all three viral diagnostics demonstrating a similar sensitivity and specificity in detecting Ross River virus and Barmah Forest virus, Australia's most common arboviruses. In summary, multi-loci metabarcoding is an affordable and efficient MX tool that enables complete mosquito-borne disease surveillance.

Sample preservation in the field and during transport can be a logistical challenge for microbiome surveys, particularly in remote areas. Sample desiccation eliminates the need for complicated cold chains and dangerous preservatives. However, the effects of desiccation on modern microbiome workflows such as gene-centric metagenomic profiling and metagenome-assembled genome (MAG) binning, remain poorly understood. In addition, most common desiccation tools such as lyophilisation cannot easily be deployed in the field. Here, we describe a proof-of-principle sample desiccator using vacuum and heat, specifically built for deployment in the field and exhibiting low power consumption and cost. We then test the effects of vacuum-heat-assisted sample desiccation followed by storage at room temperature, in comparison to conventional freezing, on multiple soil and animal faecal samples, via metagenomic and 16S rRNA amplicon sequencing. We consider multiple metrics related to the success of DNA extraction, sequencing, contig assembly, OTU clustering, gene annotation and MAG construction, as well as effects on inferred microbial community composition. We find that the impact of drying on considered success metrics was almost always either minor, non-significant or positive. For a subset of source materials we observed moderate but statistically significant differences in terms of inferred microbial taxonomic and genetic composition. We conclude that vacuum- and heat-assisted desiccation can be a useful, practical and cost-effective tool for microbiome field surveys, when a high consistency with frozen samples is not required.

Reliable tools for the identification of genetic sex are invaluable in many fields of biology, but their design requires knowledge of sex-linked sequences, which is lacking in many taxa. Restriction-site-associated DNA sequencing (RADseq) is widely used to identify sex-linked markers, but multiple distinct strategies are employed, and it is often not obvious which is most suitable. In this study, we compare two approaches for using RADseq to identify sex-linked markers. We use the common newt, Lissotriton vulgaris, as our study system, providing a challenging combination of homomorphic sex chromosomes and an exceptionally large genome. We attempt an associative approach, sequencing 60 adult newts of known-sex individuals, and compare this to a linkage mapping approach utilising a family of 146 offspring with unknown sex. After optimisation for a highly paralogous genome, the associative approach identifies five Y-chromosome-linked markers in L. vulgaris, and we design a robust PCR protocol for molecular sexing of four more related species. Via the linkage approach, we construct a high-density map featuring 10,763 markers, matching the observed karyotype of L. vulgaris and showing broad synteny with the Iberian ribbed newt (Pleurodeles waltl). However, without incorporating the markers identified via the association-based approach, we cannot confidently distinguish a sex-determining region in the linkage map, either by analysing marker density or by identifying clusters of paternal markers. We conclude that linkage mapping alone is unlikely to yield sex-linked markers in organisms with very small sex-determining regions, whereas association-based RADseq can still be effective under these conditions.

Yellow monkeyflowers (Mimulus guttatus complex, Phrymaceae) are a powerful system for studying ecological adaptation, reproductive variation, and genome evolution. To initiate pan-genomics in this group, we present four chromosome-scale assemblies and annotations of accessions spanning a broad evolutionary spectrum: two from a single M. guttatus population, one from the closely related selfing species M. nasutus, and one from a more divergent species M. tilingii. All assemblies are highly complete and resolve centromeric and repetitive regions. Comparative analyses reveal such extensive structural variation in repeat-rich, gene-poor regions that large portions of the genome are unalignable across accessions. As a result, this Mimulus pan-genome is primarily informative in genic regions, underscoring limitations of resequencing approaches in such polymorphic taxa. We document gene presence–absence, investigate the recombination landscape using high-resolution linkage data, and quantify nucleotide diversity. Surprisingly, pairwise differences at fourfold synonymous sites are exceptionally high—even in regions of very low recombination—reaching ~3.2% within a single M. guttatus population, ~7% within the interfertile M. guttatus species complex (approximately equal to SNP divergence between great apes and Old World monkeys), and ~7.4% between that complex and the reproductively isolated M. tilingii. Genome-wide patterns of nucleotide variation show little evidence of linked selection, and instead suggest that the concentration of genes (and likely selected sites) in high-recombination regions may buffer diversity loss. These assemblies, annotations, and comparative analyses provide a robust genomic foundation for Mimulus research and offer new insights into the interplay of recombination, structural variation, and molecular evolution in highly diverse plant genomes.

Inferring community composition from shotgun sequencing of environmental DNA is highly dependent on the completeness of reference databases used to assign taxonomic information as well as the pipeline used. While the number of complete, fully assembled reference genomes is increasing rapidly, their taxonomic coverage is generally too sparse to use them to build complete reference databases that span all or most of the target taxa. Low-coverage, whole genome sequencing, or skimming, provides a cost-effective and scalable alternative source of genome-wide information in the interim. Without enough coverage to assemble large contigs of nuclear DNA, much of the utility of a genome skim in the context of taxonomic annotation is found in its short read form. However, previous methods have not been able to fully leverage the data in this format. We demonstrate the utility of wholeskim, a pipeline for the indexing of k-mers present in genome skims and subsequent querying of these indices with short DNA reads. Wholeskim expands on the functionality of kmindex, a software which utilises Bloom filters to efficiently index and query billions of k-mers. Using a collection of thousands of plant genome skims, wholeskim is the only software that is able to index and query the skims in their unassembled form. It is able to correctly annotate 1.16× more simulated reads and 2.48× more true sedaDNA reads in 0.32× of the time required by Holi, another metagenomic pipeline that uses genome skims in their assembled form as its reference database input. We also explore the effects of taxonomic and genomic completeness of the reference database on the accuracy and sensitivity of read assignment. Increasing the genomic coverage of the genome skims used as reference increases the number of correctly annotated reads, but with diminishing returns after ~1× depth of coverage. Increasing taxonomic coverage clearly reduces the number of false negative taxa in the dataset, but we also demonstrate that it does not greatly impact false positive annotations.

RETRACTION: B. Mayne, T. Espinoza, D. Roberts, G. L. Butler, S. Brooks, D. Korbie and S. Jarman, “Nonlethal Age Estimation of Three Threatened Fish Species Using DNA Methylation: Australian Lungfish, Murray Cod and Mary River Cod,” Molecular Ecology Resources 21, no. 7 (2021): 2324-2332, https://doi.org/10.1111/1755-0998.13440.

The above article, published online on 23 June 2021 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editors-in-Chief, Shawn Narum and Ben Sibbett; and John Wiley & Sons Ltd. The retraction has been agreed upon following an alert from one of the authors, S. Jarman, identifying that the results shown in Figure 1 are inaccurate and do not represent a real relationship between age and DNA methylation. Consequently, the editors consider the results and conclusions invalid. The authors, S. Jarman, G. Butler and B. Mayne, agree with the decision to retract this article.

RETRACTION: B. Mayne, W. Mustin, V. Baboolal, F. Casella, K. Ballorain, M. Barret, M. A. Vanderklift, A. D. Tucker, D. Korbie, S. Jarman and O. Berry, “Age Prediction of Green Turtles With an Epigenetic Clock,” Molecular Ecology Resources 22, no. 6 (2022): 2275-2284, https://doi.org/10.1111/1755-0998.13621.

The above article, published online on 15 April 2022 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editors-in-Chief, Shawn Narum and Ben Sibbett; and John Wiley & Sons Ltd. The retraction has been agreed upon following an alert from one of the authors, S. Jarman, identifying that the results shown in Figure 2 are inaccurate and do not represent a real relationship between age and DNA methylation. Consequently, the editors consider the results and conclusions invalid. The authors, S. Jarman, B. Mayne, M. A. Vanderklift, O. Berry, K. Ballorain and M. Barret agree with the decision to retract this article.