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Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin). However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples. We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S meta-barcoding) pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart). The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease.

Structural variants (SVs) correspond to different types of genomic variants larger than 50 bp. Many findings suggest the use of long-read (LR) rather than short-read (SR) sequencing to improve the accuracy of SVs detection. Here, we present the results of an in-depth analysis for detection of SVs, mainly large insertions and deletions, in 14 French bovine breeds, based on whole-genome sequence (WGS) data comprising 176 LR and 571 SR samples, with 154 individuals having both LR and SR data available. We first investigated possible biases on the performances of well-known SVs detection tools, namely CUTESV, PBSV, and SNIFFLES, using LR from different technologies, including PacBio HiFi, Oxford ONT, and PacBio CLR. We subsequently highlighted the abilities of tools for detecting SVs (DELLY, LUMPY, and MANTA) and for genotyping known SVs (GRAPHTYPER, SVTYPER, PARAGRAPH, and VG toolkit) using SR data. We then show how the incremental composition of samples in the reference panel affected the SVs genotyping for six validation individuals sequenced in SR. We then searched for the optimal parameters and created the final SVs reference panel consisting of 25,191 deletions and 30,118 insertions. Finally, we emphasized the landscape of the genotyped SVs segregating across 571 SR individuals of 14 breeds.

Although several maize genome assemblies are publicly available, those of lines important to European breeding programs are underrepresented. Using PacBio long-read sequencing, we assembled high-quality chromosome-level genomes of 29 key lines of European breeding relevance, encompassing Northern flint and European flint lines used for adaptation to Northern European climate, lines derived from European landraces of tropical origin, and American temperate dent lines adapted to European regions. Genome assembly sizes range from 2.17 to 2.35 gigabases, with scaffold N50s ranging from 219 to 254 megabases. Completeness assessment revealed BUSCO scores ranging from 97.7 to 98.5 and merqury completeness scores ranging from 96.62 to 98.30. Calling structural variants and SNPs relative to the B73 reference sequence revealed the expected separation of inbred groups. Flint lines contribute the highest number of novel variants, thus emphasizing the importance of sequencing flint material to complete the maize pangenome. These high-quality genome assemblies therefore provide new opportunities to understand the dynamics of maize structural variation, and to identify the functional variations underlying maize phenotypic diversity.

Basic knowledge on the biology and epidemiology of equine strongylid species still needs to be improved to contribute to the design of better parasite control strategies. Nemabiome metabarcoding is a convenient tool to quantify and identify species in bulk samples that could overcome the hurdle that cyathostomin morphological identification represents. To date, this approach has relied on the internal transcribed spacer 2 (ITS-2) of the ribosomal RNA gene, with a limited investigation of its predictive performance for cyathostomin communities. Using DNA pools of single cyathostomin worms, this study aimed to provide the first elements to compare performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. Barcode predictive abilities were compared across various mock community compositions of two, five and 11 individuals from distinct species. The amplification bias of each barcode was estimated. Results were also compared between various types of biological samples, i.e ., eggs, infective larvae or adults. Bioinformatic parameters were chosen to yield the closest representation of the cyathostomin community for each barcode, underscoring the need for communities of known composition for metabarcoding purposes. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types. However, imperfect correlations were found between relative abundances from infective larvae and other life-stages for Cylicostephanus species using the ITS-2 barcode. While the results remain limited by the considered biological material, they suggest that additional improvements are needed for both the ITS-2 and COI barcodes.

AbstractBackgroundBacterial cold-water disease, which is caused by Flavobacterium psychrophilum, is one of the major diseases that affect rainbow trout (Oncorhynchus mykiss) and a primary concern for trout farming. Better knowledge of the genetic basis of resistance to F. psychrophilum would help to implement this trait in selection schemes and to investigate the immune mechanisms associated with resistance. Various studies have revealed that skin and mucus may contribute to response to infection. However, previous quantitative trait loci (QTL) studies were conducted by using injection as the route of infection. Immersion challenge, which is assumed to mimic natural infection by F. psychrophilum more closely, may reveal different defence mechanisms.ResultsTwo isogenic lines of rainbow trout with contrasting susceptibilities to F. psychrophilum were crossed to produce doubled haploid F2 progeny. Fish were infected with F. psychrophilum either by intramuscular injection (115 individuals) or by immersion (195 individuals), and genotyped for 9654 markers using RAD-sequencing. Fifteen QTL associated with resistance traits were detected and only three QTL were common between the injection and immersion. Using a model that accounted for epistatic interactions between QTL, two main types of interactions were revealed. A “compensation-like” effect was detected between several pairs of QTL for the two modes of infection. An “enhancing-like” interaction effect was detected between four pairs of QTL. Integration of the QTL results with results of a previous transcriptomic analysis of response to F. psychrophilum infection resulted in a list of potential candidate immune genes that belong to four relevant functional categories (bacterial sensors, effectors of antibacterial immunity, inflammatory factors and interferon-stimulated genes).ConclusionsThese results provide new insights into the genetic determinism of rainbow trout resistance to F. psychrophilum and confirm that some QTL with large effects are involved in this trait. For the first time, the role of epistatic interactions between resistance-associated QTL was evidenced. We found that the infection protocol used had an effect on the modulation of defence mechanisms and also identified relevant immune functional candidate genes.

Abstract The Periconia fungal genus belongs to the phylum Ascomycota, order Pleosporales, family Periconiaceae. Periconia are found in many habitats, but little is known about their ecology. Several species from this genus produce bioactive molecules. Periconia digitata extracts were shown to be deadly active against the pine wilt nematode. Furthermore, P. digitata was shown to inhibit the plant pathogenic oomycete Phytophthora parasitica . Because P. digitata has great potential as a biocontrol agent and high quality genomic resources are still lacking in the Periconiaceae family, we generated long-read genomic data for P. digitata . Using PacBio Hifi sequencing technology, we obtained a highly-contiguous genome assembled in 13 chromosomes and totaling ca. 39 Mb. In addition, we produced a reference transcriptome, based on 12 different culture conditions, and proteomic data to support the genome annotation. Besides representing a new reference genome within the Periconiaceae, this work will contribute to our better understanding of the Eukaryotic tree of life and opens new possibilities in terms of biotechnological applications.

Inspired by the production of reference data sets in the Genome in a Bottle project, we sequenced one Charolais heifer with different technologies: Illumina paired-end, Oxford Nanopore, Pacific Biosciences (HiFi and CLR), 10X Genomics linked-reads, and Hi-C. In order to generate haplotypic assemblies, we also sequenced both parents with short reads. From these data, we built two haplotyped trio high quality reference genomes and a consensus assembly, using up-to-date software packages. The assemblies obtained using PacBio HiFi reaches a size of 3.2 Gb, which is significantly larger than the 2.7 Gb ARS-UCD1.2 reference. The BUSCO score of the consensus assembly reaches a completeness of 95.8%, among highly conserved mammal genes. We also identified 35,866 structural variants larger than 50 base pairs. This assembly is a contribution to the bovine pangenome for the “Charolais” breed. These datasets will prove to be useful resources enabling the community to gain additional insight on sequencing technologies for applications such as SNP, indel or structural variant calling, and de novo assembly.

CpG methylation, a key epigenetic mark involved in gene regulation, development, and other biological processes, is commonly analyzed using Whole-Genome Bisulfite Sequencing (WGBS). However, bisulfite treatment causes significant DNA degradation. Enzymatic Methyl-seq (EM-seq) offers a short-read alternative that preserves DNA integrity but requires conversion steps, limiting its compatibility with downstream analyses. Third-generation sequencing technologies, such as Oxford Nanopore Technologies (ONT) and PacBio, enable direct detection of DNA modifications without altering the DNA, providing simultaneous genome and epigenome information. This work presents a comprehensive dataset combining long- and short-read sequencing data, including ONT, PacBio, Enzymatic Methyl-seq, and WGBS, for two agronomically relevant species: pig (Sus scrofa) and quail (Coturnix japonica). Data quality evaluation reveals high nucleotide quality scores for PacBio and short reads, robust alignment rates for long reads, and inter-method correlations in CpG methylation calling ranging from 0.76 to 0.99. This dataset is a valuable resource for training methylation callers and represents the first combined methylation dataset for these species, providing an essential benchmark for assessing emerging sequencing technologies.

After publication of this work [1], we noted that there was an error in Table 3 Line 4.

Pseudo-homothallism is a reproductive strategy elected by some fungi producing heterokaryotic sexual spores containing genetically different but sexually compatible nuclei. This lifestyle appears as a compromise between true homothallism (self-fertility with predominant inbreeding) and complete heterothallism (with exclusive outcrossing). However, pseudohomothallic species face the problem of maintaining heterokaryotic mycelia to fully benefit from this lifestyle, as homokaryons are self-sterile. Here, we report on the structure of chromosome 1 in mat+ and mat− isolates of strain S of the pseudohomothallic fungus Podospora anserina. Chromosome 1 contains either one of the mat+ and mat− mating types of P. anserina, which is mostly found in nature as a mat+/mat− heterokaryotic mycelium harboring sexually compatible nuclei. We identified a “mat” region ∼0.8 Mb long, devoid of meiotic recombination and containing the mating-type idiomorphs, which is a candidate to be involved in the maintenance of the heterokaryotic state, since the S mat+ and S mat− strains have different physiology that may enable hybrid-vigor-like phenomena in the heterokaryons. The mat region contains 229 coding sequences. A total of 687 polymorphisms were detected between the S mat+ and S mat− chromosomes. Importantly, the mat region is colinear between both chromosomes, which calls for an original mechanism of recombination inhibition. Microarray analyses revealed that 10% of the P. anserina genes have different transcriptional profiles in S mat+ and S mat−, in line with their different phenotypes. Finally, we show that the heterokaryotic state is faithfully maintained during mycelium growth of P. anserina, yet mat+/mat+ and mat−/mat− heterokaryons are as stable as mat+/mat− ones, evidencing a maintenance of heterokaryosis that does not rely on fitness-enhancing complementation between the S mat+ and S mat− strains.