Machine-Learning-Based Single-Molecule Quantification of Circulating MicroRNA Mixtures.

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression and are emerging as powerful indicators of diseases. MiRs are secreted in blood plasma and thus may report on systemic aberrations at an early stage via liquid biopsy analysis. We present a method for multiplexed single-molecule detection and quantification of a selected panel of miRs. The proposed assay does not depend on sequencing, requires less than 1 mL of blood, and provides fast results by direct analysis of native, unamplified miRs. This is enabled by a novel combination of compact spectral imaging and a machine learning-based detection scheme that allows simultaneous multiplexed classification of multiple miR targets per sample. The proposed end-to-end pipeline is extremely time efficient and cost-effective. We benchmark our method with synthetic mixtures of three target miRs, showcasing the ability to quantify and distinguish subtle ratio changes between miR targets.

Single-molecule approaches for DNA damage detection and repair: A focus on Repair Assisted Damage Detection (RADD).

The human genome is continually exposed to various stressors, which can result in DNA damage, mutations, and diseases. Among the different types of DNA damage, single-strand lesions are commonly induced by external stressors and metabolic processes. Accurate detection and quantification of DNA damage are crucial for understanding repair mechanisms, assessing environmental impacts, and evaluating response to therapy. However, traditional techniques have limitations in sensitivity and the ability to detect multiple types of damage. In recent years, single-molecule fluorescence approaches have emerged as powerful tools for precisely localizing and quantifying DNA damage. Repair Assisted Damage Detection (RADD) is a single-molecule technique that employs specific repair enzymes to excise damaged bases and incorporates fluorescently labeled nucleotides to visualize the damage. This technique provides valuable insights into repair efficiency and sequence-specific damage. In this review, we discuss the principles and applications of RADD assays, highlighting their potential for enhancing our understanding of DNA damage and repair processes.

Genome sequences of three Aegilops species of the section Sitopsis reveal phylogenetic relationships and provide resources for wheat improvement.

Aegilops is a close relative of wheat (Triticum spp.), and Aegilops species in the section Sitopsis represent a rich reservoir of genetic diversity for the improvement of wheat. To understand their diversity and advance their utilization, we produced whole-genome assemblies of Aegilops longissima and Aegilops speltoides. Whole-genome comparative analysis, along with the recently sequenced Aegilops sharonensis genome, showed that the Ae. longissima and Ae. sharonensis genomes are highly similar and are most closely related to the wheat D subgenome. By contrast, the Ae. speltoides genome is more closely related to the B subgenome. Haplotype block analysis supported the idea that Ae. speltoides genome is closest to the wheat B subgenome, and highlighted variable and similar genomic regions between the three Aegilops species and wheat. Genome-wide analysis of nucleotide-binding leucine-rich repeat (NLR) genes revealed species-specific and lineage-specific NLR genes and variants, demonstrating the potential of Aegilops genomes for wheat improvement.

Multiple wheat genomes reveal global variation in modern breeding.

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars.

Durum wheat genome highlights past domestication signatures and future improvement targets.

The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement.

Wild emmer genome architecture and diversity elucidate wheat evolution and domestication.

Wheat (Triticum spp.) is one of the founder crops that likely drove the Neolithic transition to sedentary agrarian societies in the Fertile Crescent more than 10,000 years ago. Identifying genetic modifications underlying wheat's domestication requires knowledge about the genome of its allo-tetraploid progenitor, wild emmer (T. turgidum ssp. dicoccoides). We report a 10.1-gigabase assembly of the 14 chromosomes of wild tetraploid wheat, as well as analyses of gene content, genome architecture, and genetic diversity. With this fully assembled polyploid wheat genome, we identified the causal mutations in Brittle Rachis 1 (TtBtr1) genes controlling shattering, a key domestication trait. A study of genomic diversity among wild and domesticated accessions revealed genomic regions bearing the signature of selection under domestication. This reference assembly will serve as a resource for accelerating the genome-assisted improvement of modern wheat varieties.