Scientific highlights

Morabine grasshoppers in the Vandiemenella viatica species group, which show karyotype diversity, have been studied for their ecological distribution and speciation in relation to their genetic and chromosomal diversity. They are good models for studying sex chromosome evolution as "old" and newly emerged sex chromosomes co-exist within the group. Here we present a reference genome for the viatica19 chromosomal race, that possesses the ancestral karyotype within the group. Using PacBio HiFi and Hi-C sequencing, we generated a chromosome-level assembly of 4.09 Gb in span, scaffold N50 of 429 Mb, and complete BUSCO score of 98.1%, containing 10 pseudo-chromosomes. We provide Illumina datasets of males and females, used to identify the X chromosome. The assembly contains 19,034 predicted protein-coding genes, and a total of 75.21% of repetitive DNA sequences. By leveraging HiFi reads, we mapped the genome-wide distribution of methylated bases (5mC and 6 mA). This comprehensive assembly offers a robust reference for morabine grasshoppers and supports further research into speciation and sex chromosome diversification within the group and its related species.

Progress in genome sequencing now enables the large-scale generation of reference genomes. Various international initiatives aim to generate reference genomes representing global biodiversity. These genomes provide unique insights into genomic diversity and architecture, thereby enabling comprehensive analyses of population and functional genomics, and are expected to revolutionize conservation genomics.

The most frequent Y-chromosomal (chrY) haplogroups in northern and eastern Europe (NEE) are well-known and thoroughly characterised. Yet a considerable number of men in every population carry rare paternal lineages with estimated frequencies around 5%. So far, limited sample-sizes and insufficient resolution of genotyping have obstructed a truly comprehensive look into the variety of rare paternal lineages segregating within populations and potential signals of population history that such lineages might convey. Here we harness the power of massive re-sequencing of human Y chromosomes to identify previously unknown population-specific clusters among rare paternal lineages in NEE. We construct dated phylogenies for haplogroups E2-M215, J2-M172, G-M201 and Q-M242 on the basis of 421 (of them 282 novel) high-coverage chrY sequences collected from large-scale databases focusing on populations of NEE. Within these otherwise rare haplogroups we disclose lineages that began to radiate ~1-3 thousand years ago in Estonia and Sweden and reveal male phylogenetic patterns testifying of comparatively recent local demographic expansions. Conversely, haplogroup Q lineages bear evidence of ancient Siberian influence lingering in the modern paternal gene pool of northern Europe. We assess the possible direction of influx of ancestral carriers for some of these male lineages. In addition, we demonstrate the congruency of paternal haplogroup composition of our dataset with two independent population-based cohorts from Estonia and Sweden.

One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro. The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites. Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.

Invasive cervical cancer (ICC) with very low titer of high-risk human papillomavirus (HPV) has worse clinical outcome than cases with high titer, indicating a difference in molecular etiology. Fresh-frozen ICC tumors (n = 49) were classified into high- and low-HPV-titer cases using real-time PCR-based HPV genotyping. The mutation spectra were studied using the AmpliSeq Comprehensive Cancer Panel and the expression profiles using total RNA sequencing, and the results were validated using the AmpliSeq Transcriptome assay. HPV DNA genotyping and RNA sequencing showed that 16.6% of ICC tumors contained very low levels of HPV DNA and HPV transcripts. Tumors with low HPV levels had more mutations with a high allele frequency and fewer mutations with low allele frequency relative to tumors with high HPV titer. A number of genes showed significant expression differences between HPV titer groups, including genes with somatic mutations. Gene ontology and pathway analyses implicated the enrichment of genes involved in DNA replication, cell cycle control and extracellular matrix in tumors with low HPV titer. The results indicate that in low titer tumors, HPVs act as trigger of cancer development whereas somatic mutations are clonally selected and become drivers of the tumor development process. In contrast, in tumors with high HPV titer the expression of HPV oncoproteins plays a major role in tumor development and the many low frequency somatic mutations represent passengers. This putative subdivision of invasive cervical tumors may explain the higher radiosensitivity of ICC tumors with high HPV titer and thereby have consequences for clinical management.

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with single molecule, real-time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods.

We assessed the diversity and composition of fungal communities in different functional tissues and the rhizosphere soil of Pinus sylvestris and Picea abies stands along the latitudinal gradient of these tree species distributions in Europe to model possible changes in fungal communities imposed by climate change. For each tree species, living needles, shoots, roots, and the rhizosphere soil were sampled and subjected to high-throughput sequencing. Results showed that the latitude and the host tree species had a limited effect on the diversity and composition of fungal communities, which were largely explained by the environmental variables of each site and the substrate they colonize. The mean annual temperature and mean annual precipitation had a strong effect on root fungal communities, isothermality on needle fungal communities, mean temperature of the warmest quarter and precipitation of the driest month on shoot fungal communities, and precipitation seasonality on soil fungal communities. Fungal communities of both tree species are predicted to shift to habitats with a lower annual temperature amplitude and with increasing precipitation during the driest month, but the suitability of these habitats as compared to the present conditions is predicted to decrease in the future.

The plant- and soil-associated microbial communities are critical to plant health and their resilience to stressors, such as drought, pathogens, and pest outbreaks. A better understanding of the structure of microbial communities and how they are affected by different environmental factors is needed to predict and manage ecosystem responses to climate change. In this study, we carried out a country-wide analysis of fungal communities associated with Pinus sylvestris growing under different environmental conditions. Needle, shoot, root, mineral, and organic soil samples were collected at 30 sites. By interconnecting the high-throughput sequencing data, environmental variables, and soil chemical properties, we were able to identify key factors that drive the diversity and composition of fungal communities associated with P. sylvestris. The fungal species richness and community composition were also found to be highly dependent on the site and the substrate they colonize. The results demonstrated that different functional tissues and the rhizosphere soil of P. sylvestris are associated with diverse fungal communities, which are driven by a combination of climatic (temperature and precipitation) and edaphic factors (soil pH), and stand characteristics.

The global RNA-binding protein ProQ has emerged as a central player in post-transcriptional regulatory networks in bacteria. While the N-terminal domain (NTD) of ProQ harbors the major RNA-binding activity, the role of the ProQ C-terminal domain (CTD) has remained unclear. Here, we have applied saturation mutagenesis coupled to phenotypic sorting and long-read sequencing to chart the regulatory capacity of Salmonella ProQ. Parallel monitoring of thousands of ProQ mutants allowed mapping of critical residues in both the NTD and the CTD, while the linker separating these domains was tolerant to mutations. Single amino acid substitutions in the NTD associated with abolished regulatory capacity strongly align with RNA-binding deficiency. An observed cellular instability of ProQ associated with mutations in the NTD suggests that interaction with RNA protects ProQ from degradation. Mutation of conserved CTD residues led to overstabilization of RNA targets and rendered ProQ inert in regulation, without affecting protein stability in vivo. Furthermore, ProQ lacking the CTD, although binding competent, failed to protect an mRNA target from degradation. Together, our data provide a comprehensive overview of residues important for ProQ-dependent regulation and reveal an essential role for the enigmatic ProQ CTD in gene regulation.

Leaves interact with a wealth of microorganisms. Among these, fungi are highly diverse and are known to contribute to plant health, leaf senescence and early decomposition. However, patterns and drivers of the seasonal dynamics of foliar fungal communities are poorly understood. We used a multifactorial experiment to investigate the influence of warming and tree genotype on the foliar fungal community on the pedunculate oak Quercus robur across one growing season. Fungal species richness increased, evenness tended to decrease, and community composition strongly shifted during the growing season. Yeasts increased in relative abundance as the season progressed, while putative fungal pathogens decreased. Warming decreased species richness, reduced evenness and changed community composition, especially at the end of the growing season. Warming also negatively affected putative fungal pathogens. We only detected a minor imprint of tree genotype and warming × genotype interactions on species richness and community composition. Overall, our findings demonstrate that warming plays a larger role than plant genotype in shaping the seasonal dynamics of the foliar fungal community on oak. These warming-induced shifts in the foliar fungal community may have a pronounced impact on plant health, plant-fungal interactions and ecosystem functions.

Thermokarst activity at permafrost sites releases considerable amounts of ancient carbon to the atmosphere. A large part of this carbon is released via thermokarst ponds, and fungi could be an important organismal group enabling its recycling. However, our knowledge about aquatic fungi in thermokarstic systems is extremely limited. In this study, we collected samples from five permafrost sites distributed across circumpolar Arctic and representing different stages of permafrost integrity. Surface water samples were taken from the ponds and, additionally, for most of the ponds also the detritus and sediment samples were taken. All the samples were extracted for total DNA, which was then amplified for the fungal ITS2 region of the ribosomal genes. These amplicons were sequenced using PacBio technology. Water samples were also collected to analyze the chemical conditions in the ponds, including nutrient status and the quality and quantity of dissolved organic carbon. This dataset gives a unique overview of the impact of the thawing permafrost on fungal communities and their potential role on carbon recycling.

The genus Passiflora comprises a large group of plants popularly known as passionfruit, much appreciated for their exotic flowers and edible fruits. The species (∼500) are morphologically variable (e.g., growth habit, size, and color of flowers) and are adapted to distinct tropical ecosystems. In this study, we generated the genome of the wild diploid species Passiflora organensis Gardner by adopting a hybrid assembly approach. Passiflora organensis has a small genome of 259 Mbp and a heterozygosity rate of 81%, consistent with its reproductive system. Most of the genome sequences could be integrated into its chromosomes with cytogenomic markers (satellite DNA) as references. The repeated sequences accounted for 58.55% of the total DNA analyzed, and the Tekay lineage was the prevalent retrotransposon. In total, 25,327 coding genes were predicted. Passiflora organensis retains 5,609 singletons and 15,671 gene families. We focused on the genes potentially involved in the locus determining self-incompatibility and the MADS-box gene family, allowing us to infer expansions and contractions within specific subfamilies. Finally, we recovered the organellar DNA. Structural rearrangements and two mitoviruses, besides relics of other mobile elements, were found in the chloroplast and mt-DNA molecules, respectively. This study presents the first draft genome assembly of a wild Passiflora species, providing a valuable sequence resource for genomic and evolutionary studies on the genus, and support for breeding cropped passionfruit species.

Insulin secretion is impaired with increasing age. In this study, we aimed to determine whether aging induces specific transcriptional changes in human islets. Laser capture microdissection was used to extract pancreatic islet tissue from 37 deceased organ donors aged 1-81 years. The transcriptomes of the extracted islets were analysed using Ion AmpliSeq sequencing. 346 genes that co-vary significantly with age were found. There was an increased transcription of genes linked to senescence, and several aspects of the cell cycle machinery were downregulated with increasing age. We detected numerous genes not linked to aging in previous studies likely because earlier studies analysed islet cells isolated by enzymatic digestion which might affect the islet transcriptome. Among the novel genes demonstrated to correlate with age, we found an upregulation of SPP1 encoding osteopontin. In beta cells, osteopontin has been seen to be protective against both cytotoxicity and hyperglycaemia. In summary, we present a transcriptional profile of aging in human islets and identify genes that could affect disease course in diabetes.

Bacteria colonize stallion semen during collection and processing which may cause disease in inseminated females or negatively affect sperm quality during storage prior to insemination. Antibiotics are added to semen extenders to control the growth of these bacteria but may induce antimicrobial resistance. Research into alternatives to antibiotics for this purpose requires knowledge of which bacteria are present in semen. Not all bacteria in semen, however, can be identified by conventional microbiological techniques. The objectives of the study were to: i) determine which bacteria are present in stallion semen using metagenomics; and ii) investigate individual differences in bacterial content in semen from all stallions on one premises. Bacterial DNA was extracted from ejaculates from seven stallions (one ejaculate per stallion) and bacteria were identified using 16S sequencing. In total, 83 bacterial genera were identified, varying from 25 to 52 among different individuals. There was a negative correlation (r = -0.81212; P < 0.05) between the presence of Treponema spp. and Advenella spp. In conclusion, most of the bacteria present in stallion semen could be identified to genus level by 16S sequencing even when present at a low frequency. This method of identification may help to clarify individual variation in bacterial content and its potential effects on fertility.

Canine respiratory coronavirus (CRCoV) has been identified as a causative agent of canine infectious respiratory disease, an upper respiratory infection affecting dogs. The epidemiology is currently opaque, with an unclear understanding of global prevalence, pathology, and genetic characteristics. In this study, Swedish privately-owned dogs with characteristic signs of canine infectious respiratory disease (n = 88) were screened for CRCoV and 13 positive samples (14.7%, 8.4-23.7% [95% confidence interval (CI)]) were further sequenced. Sequenced Swedish CRCoV isolates were highly similar despite being detected in dogs living in geographically distant locations and sampled across 3 years (2013-2015). This is due to a single introduction into Swedish dogs in approximately 2010, as inferred by time structured phylogeny. Unlike other CRCoVs, there was no evidence of recombination in Swedish CRCoV viruses, further supporting a single introduction. Finally, there were low levels of polymorphisms, in the spike genes. Overall, we demonstrate that there is little diversity of CRCoV which is endemic in Swedish dogs.