Molecular Ecology Resources最新文献

Genotype imputation, the inference of missing genotypes using a reference set of population haplotypes, is a cost-effective tool for improving the quality and quantity of genetic datasets. Imputation is usually applied to large and well-characterised datasets of humans and livestock, even though it could also benefit smaller natural populations. This study aims to understand the best practices and effectiveness of imputation with a small reference panel for species with low genetic diversity, using a case study of a population of the hihi/stitchbird (Notiomystis cincta). We used a leave-one-out method to test imputation on 30 high-coverage hihi individuals where SNPs were masked before being imputed with Beagle v5.4. Imputation accuracy was measured using r², the correlation between imputed and ground truth genotype dosages. We tested combinations of five imputation parameters, the inclusion of two linkage maps, reference panels of different sizes and compositions and targets of various SNP densities and sporadic missingness. We achieved mean r² exceeding 0.95 in most tests from a small reference panel of high-fecundity individuals. Imputation accuracy was not improved by including a linkage map and decreased at very low SNP densities. Imputed SNPs were filtered using r² to assess downstream heterozygosity calculations, the site frequency spectrum (SFS) and inference of runs of homozygosity (ROHs). We found that filtering and SNP density greatly affected heterozygosity and SFS at low SNP densities but that ROH inference was relatively robust to both. We provide a template for testing and optimising imputation in other wild populations.

DNA barcoding traditionally relies on Sanger sequencing but faces limitations with degraded samples. High-throughput sequencing (HTS) offers a cost-effective alternative, enabling rapid barcode generation for extensive datasets. The advantage of HTS is its ability to employ multiplexing strategies, allowing thousands of samples to be processed simultaneously in a single sequencing run. This study presents the CODEX approach, a double-indexed HTS method designed to sequence overlapping cox-1 barcode fragments, suitable for samples with degraded DNA. The approach was applied to neogastropods, a diverse lineage of marine molluscs, using specimens (both recently collected and relatively older) from the Muséum national d'Histoire naturelle (MNHN) collections. The pipeline was used to process 15,076 samples, yielding 10,905 cleaned and assembled sequences, achieving a success rate of 72.33%. The CODEX method demonstrated advantages over Sanger sequencing by enabling the recovery of barcodes from samples previously deemed unsuitable, with significantly reduced costs (€0.5 per sequence vs. €4.5). Notably, DNA quality and sequencing success were strongly correlated with collection date, emphasising the impact of preservation methods and storage conditions. Sequencing success rates varied among families but were not correlated with phylogenetic relationships or specimen size, indicating the robustness of the primers designed for neogastropods. This study highlights the efficiency of the CODEX approach for large-scale DNA barcoding projects, especially when handling degraded samples. The CODEX pipeline and associated resources are publicly accessible, offering a scalable solution for molecular systematics and beyond.

Molecular tools are increasingly being used to survey the presence of biodiversity and their interactions within ecosystems. Indirect methods, like environmental DNA (eDNA) and invertebrate-derived DNA (iDNA), are dependent on sequence databases with accurate and sufficient taxonomic representation. These methods are increasingly being used in regions and habitats where direct detection or observations can be difficult for a variety of reasons. Madagascar is a biodiversity hotspot with a high proportion of endemic species, many of which are threatened or endangered. Here we describe a new resource, VoronaGasyCodes, a curated database of newly published genetic sequences from Malagasy birds. Our database is currently populated with six mitochondrial genes or DNA barcodes for 142 species including 70% of the birds endemic to the island and will be periodically updated as new data become available. We demonstrate the utility of our database with an iDNA study of leech blood meals where we successfully identified 77% of the hosts to species. These types of resources for characterising biodiversity are critical for insights into species distribution, discovery of new taxa, novel ecological connections and advancing conservation and restoration measures.

DNA methylation is crucial for genome regulation and provides key insights into the interaction between genetics and environmental factors, offering valuable perspectives for ecological research. However, knowledge of DNA methylation patterns in nonmodel invertebrates remains limited. The present study addresses this knowledge gap by conducting the first methylome profiling of the Pacific crown-of-thorns seastar (CoTS; Acanthaster cf. solaris), a coral-eating species that aggravates the decline of Indo-Pacific coral reefs. Using Oxford Nanopore Technology (ONT) we generated long-read sequences, covering over 90% of CpG dinucleotides in the CoTS genome. Our analysis revealed a mosaic methylation landscape with moderate genome-wide methylation levels of 37.7%. Comparative analysis highlights the intermediate methylation state observed in other deuterostome invertebrates, positioning them between the hypomethylated genomes of protostomes and the hypermethylated genomes of vertebrates. Methylation in CoTS was predominantly localised within gene bodies, especially in intronic regions, enabling modulation of gene expression and potentially supporting fitness in dynamic marine environments. Additionally, elevated methylation in repetitive elements suggests a role in genome defence. This study demonstrates the effectiveness of ONT for comprehensive methylome analysis in ecologically important nonmodel species and deepens our understanding of the epigenetic landscape in deuterostome invertebrates. We also present a detailed laboratory and bioinformatics workflow, including modified phenol–chloroform protocols that address the challenge of extracting high molecular weight DNA from marine invertebrates. Together with the methylome profiles, these resources serve as a foundation for future research, enabling investigations into DNA methylation functions, applications for CoTS outbreak management and comparative studies across diverse lineages.

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.