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Bovine leukemia virus (BLV) infects cattle and integrates into host DNA, causing enzootic bovine leukosis (EBL), an aggressive B-cell lymphoma. Here, we developed a novel proviral DNA-capture sequencing (proviral DNA-capture-seq) method investigating BLV proviral integration in two B-cell lymphoma lines, BLSC-KU1 and BLSC-KU17, derived from BLV-infected cattle with EBL. We designed BLV-specific biotinylated probes to capture the provirus genome and enrich libraries for next-generation sequencing. Validation showed high specificity and efficient enrichment of target sequence reads as well as identification of three BLV proviral integration sites on BLV persistently infected FLK-BLV cells as a positive control. We successfully detected a single BLV proviral integration site on chromosome 19 of BLSC-KU1 and chromosome 9 of BLSC-KU17, which were confirmed by standard PCR and Sanger sequencing. Further, a defective provirus in BLSC-KU1 and complete BLV proviral sequence in BLSC-KU17 were confirmed using long PCR and sequencing. This is the first study to provide comprehensive information on BLV proviral structure and viral integration in BLSC-KU1 and BLSC-KU17. Moreover, the proposed method can facilitate understanding of the detailed mechanisms underlying BLV-induced leukemogenesis and may be used as an innovative tool to screen BLV-infected cattle at risk at an earlier stage than those that have already developed lymphoma.

An understanding of how biotic interactions shape species’ distributions is central to predicting host–symbiont responses under climate change. Switches to locally adapted algae have been proposed to be an adaptive strategy of lichen-forming fungi to cope with environmental change. However, it is unclear how lichen photobionts respond to environmental gradients, and whether they play a role in determining the fungal host’s upper and lower elevational limits. Deep-coverage Illumina DNA metabarcoding was used to track changes in the community composition of Trebouxia algae associated with two phylogenetically closely related, but ecologically divergent fungal hosts along a steep altitudinal gradient in the Mediterranean region. We detected the presence of multiple Trebouxia species in the majority of thalli. Both altitude and host genetic identity were strong predictors of photobiont community assembly in these two species. The predominantly clonally dispersing fungus showed stronger altitudinal structuring of photobiont communities than the sexually reproducing host. Elevation ranges of the host were not limited by the lack of compatible photobionts. Our study sheds light on the processes guiding the formation and distribution of specific fungal–algal combinations in the lichen symbiosis. The effect of environmental filtering acting on both symbiotic partners appears to shape the distribution of lichens.

Background Liquid biopsy has emerged as a non‐invasive method for real‐time cancer monitoring especially in genitourinary (GU) oncology. Most current studies utilize a panel‐based molecular profiling ranging from 50–600 genes; however, a comprehensive exome‐wide profiling of real‐world patient samples has been lacking. Methods Over 2000 liquid biopsy samples were analyzed in this study, including urine samples from early‐stage bladder cancer and plasma samples from prostate, lung, breast, esophageal, head and neck cancers, among others. Cell‐free DNA (cfDNA) was extracted from these samples and analyzed using PredicineWES+™, a boosted comprehensive whole‐exome sequencing (WES) assay with an in‐depth coverage of 600 cancer‐related genes derived from the PredicineATLAS™ panel. Data analysis was conducted in‐house using Predicine's DeepSea bioinformatics software. Results The PredicineWES+™ assay demonstrated high sensitivity for detecting somatic mutations across the exome and showed comparable tumor mutational burden (TMB) estimates with the PredicineATLAS™ panel. Interestingly, the highest tumor TMB score was observed in bladder cancer among the analyzed cancers, which is consistent with literature using tissue‐based genomic profiling. The most common cancer variants include TP53, ERBB2, KRAS, PIK3CA, FGFR3, APC, among others. Conclusion Liquid biopsy‐based genomic profiling across various cancer types provides an in‐depth analysis of biomarker discovery for personalized cancer care, setting the foundation for improved cancer diagnosis and personalized treatment strategies for urological diseases.

Extending the durability of plant resistance genes towards fungal pathogens is a major challenge. We identified and investigated the relationship between two avirulence genes of Leptosphaeria maculans, AvrLm3 and AvrLm4‐7. When an isolate possesses both genes, the Rlm3‐mediated resistance of oilseed rape (Brassica napus) is not expressed due to the presence of AvrLm4‐7 but virulent isolates toward Rlm7 recover the AvrLm3 phenotype.Combining genetic and genomic approaches (genetic mapping, RNA‐seq, BAC (bacterial artificial chromosome) clone sequencing and de novo assembly) we cloned AvrLm3, a telomeric avirulence gene of L. maculans. AvrLm3 is located in a gap of the L. maculans reference genome assembly, is surrounded by repeated elements, encodes for a small secreted cysteine‐rich protein and is highly expressed at early infection stages.Complementation and silencing assays validated the masking effect of AvrLm4‐7 on AvrLm3 recognition by Rlm3 and we showed that the presence of AvrLm4‐7 does not impede AvrLm3 expression in planta. Y2H assays suggest the absence of physical interaction between the two avirulence proteins.This unusual interaction is the basis for field experiments aiming to evaluate strategies that increase Rlm7 durability.

Microsatellites, or simple sequence repeats (SSRs), have long played a major role in genetic studies due to their typically high polymorphism. They have diverse applications, including genome mapping, forensics, ascertaining parentage, population and conservation genetics, identification of the parentage of polyploids, and phylogeography. We compare SSRs and newer methods, such as genotyping by sequencing (GBS) and restriction site associated DNA sequencing (RAD-Seq), and offer recommendations for researchers considering which genetic markers to use. We also review the variety of techniques currently used for identifying microsatellite loci and developing primers, with a particular focus on those that make use of next-generation sequencing (NGS). Additionally, we review software for microsatellite development and report on an experiment to assess the utility of currently available software for SSR development. Finally, we discuss the future of microsatellites and make recommendations for researchers preparing to use microsatellites. We argue that microsatellites still have an important place in the genomic age as they remain effective and cost-efficient markers.

Somatic multigene analysis by next‐generation sequencing (NGS) is routinely integrated in medical oncology for clinical decision‐making. However, with the fast‐growing number of recommended and required genes as well as pan‐cancer biomarkers, small panels have become vastly insufficient. Comprehensive genomic profiling (CGP) is, thus, required to screen for clinically relevant markers. In this multicentric study, we report on an extensive analysis across seven centers comparing the results of the novel OncoDEEP CGP assay with the diagnostically validated TruSight Oncology 500 (TSO500) kit on 250 samples. Overall concordance was 90% for clinically relevant gene variants and >96% for more complex biomarkers. Agreement for fusion detection was 94% for the 11 overlapping clinically actionable driver genes. The higher coverage uniformity of OncoDEEP compared to TSO500 allows users to pool more samples per sequencing run. Tertiary data analysis, including reporting, is integrated in the OncoDEEP solution, whereas this is an add‐on for TSO500. Finally, we showed that, analytically, the OncoDEEP panel performs well, thereby advocating its use for CGP of solid tumors in diagnostic laboratories, providing an all‐in‐one solution for optimal patient management. In molecular cancer diagnostics, comprehensive genomic profiling (CGP) is going to replace the small NGS panels since it provides all clinically relevant somatic variants as well as genomic biomarkers with clinical value. Here, we compared two CGP assays and demonstrate that the choice for diagnostic implementation will depend on the specific requirements of the laboratory because each assay has its pros and cons.

INTRODUCTION Studies have correlated living close to major roads with Alzheimer's disease (AD) risk. However, the mechanisms responsible for this link remain unclear. METHODS We exposed olfactory mucosa (OM) cells of healthy individuals and AD patients to diesel emissions (DE). Cytotoxicity of exposure was assessed, mRNA, miRNA expression, and DNA methylation analyses were performed. The discovered altered pathways were validated using data from the human population‐based Rotterdam Study. RESULTS DE exposure resulted in an almost four‐fold higher response in AD OM cells, indicating increased susceptibility to DE effects. Methylation analysis detected different DNA methylation patterns, revealing new exposure targets. Findings were validated by analyzing data from the Rotterdam Study cohort and demonstrated a key role of nuclear factor erythroid 2–related factor 2 signaling in responses to air pollutants. DISCUSSION This study identifies air pollution exposure biomarkers and pinpoints key pathways activated by exposure. The data suggest that AD individuals may face heightened risks due to impaired cellular defenses. Highlights Healthy and AD olfactory cells respond differently to DE exposure. AD cells are highly susceptible to DE exposure. The NRF2 oxidative stress response is highly activated upon air pollution exposure. DE‐exposed AD cells activate the unfolded protein response pathway. Key findings are also confirmed in a population‐based study.

This article is a Commentary on Meinke, 226: 306–325.

Premise of the study: Microsatellite primers were developed for Pedicularis ishidoyana (Orobanchaceae), an endangered and hemiparasitic plant that is narrowly endemic to Korea. Because its populations are threatened by loss of suitable habitat, conservation efforts are required. Methods and Results: We developed polymorphic microsatellite loci through reference mapping of 300-bp paired-end reads obtained from Illumina MiSeq data. In all, 74 primer pairs were designed and 32 were amplified. Of these, 18 pairs were polymorphic, with two to six alleles each occurring in 26 individual plants. Expected and observed heterozygosities ranged from 0.142 to 0.703 and from 0.077 to 0.615, respectively. Conclusions: These microsatellite markers are expected to be useful for studies of the population genetics of P. ishidoyana.

The application of next generation sequencing (NGS) for the analysis of mitochondrial (mt) DNA, short tandem repeats (STRs), and single nucleotide polymorphism (SNPs) has demonstrated great promise for challenging forensic specimens, such as degraded, limited, and mixed samples. Target enrichment using probe capture rather than PCR amplification offers advantages for analysis of degraded DNA since two intact PCR primer sites in the template DNA molecule are not required. Furthermore, NGS software programs can help remove PCR duplicates to determine initial template copy numbers of a shotgun library. Moreover, the same shotgun library prepared from a limited DNA source can be enriched for mtDNA as well as nuclear markers by hybrid capture with the relevant probe panels. Here, we demonstrate the use of this strategy in the analysis of limited and mock degraded samples using our custom probe capture panels for massively parallel sequencing of the whole mtgenome and 426 SNP markers. We also applied the mtgenome capture panel in a mixed sample and analyzed using both phylogenetic and variant frequency based bioinformatics tools to resolve the minor and major contributors. Finally, the results obtained on individual telogen hairs demonstrate the potential of probe capture NGS analysis for both mtDNA and nuclear SNPs for challenging forensic specimens.