A comprehensive assessment of exome capture methods for RNA sequencing of formalin-fixed and paraffin-embedded samples.

RNA-Seq analysis of Formalin-Fixed and Paraffin-Embedded (FFPE) samples has emerged as a highly effective approach and is increasingly being used in clinical research and drug development. However, the processing and storage of FFPE samples are known to cause extensive degradation of RNAs, which limits the discovery of gene expression or gene fusion-based biomarkers using RNA sequencing, particularly methods reliant on Poly(A) enrichment. Recently, researchers have developed an exome targeted RNA-Seq methodology that utilizes biotinylated oligonucleotide probes to enrich RNA transcripts of interest, which could overcome these limitations. Nevertheless, the standardization of this experimental framework, including probe designs, sample multiplexing, sequencing read length, and bioinformatic pipelines, remains an essential requirement. In this study, we conducted a comprehensive comparison of three main commercially available exome capture kits and evaluated key experimental parameters, to provide the overview of the advantages and limitations associated with the selection of library preparation protocols and sequencing platforms. The results provide valuable insights into the best practices for obtaining high-quality data from FFPE samples.

Single-cell RNA-seq of Lotus japonicus provide insights into identification and function of root cell types of legume.

The roots of legume plant play a crucial role in nitrogen fixation. However, the transcriptomes of different cell types of legume root and their functions remain largely unknown. Here, we performed single-cell RNA sequencing and profiled more than 22,000 single cells from root tips of Lotus japonicus, a model species of legume. We identified seven clusters corresponding to seven major cell types, which were validated by in situ hybridization. Further analysis revealed regulatory programs including phytohormone and nodulation associated with specific cell types, and revealed conserved and diverged features for the cell types. Our results represent the first single-cell resolution transcriptome for legume root tips and a valuable resource for studying the developmental and physiological functions of various cell types in legumes.

Resequencing of 296 cultivated and wild lotus accessions unravels its evolution and breeding history.

Lotus (family: Nelumbonaceae) are perennial aquatic plants that represent one of the most ancient basal dicots. In the present study, we resequenced 296 lotus accessions from various geographical locations and germplasms to explore their genomic diversity and population structure. This germplasm set consisted of four accessions of American wild lotus and 292 accessions of Asian lotus, which were divided into four subgroups: wild, rhizome, flower and seed. Total single nucleotide polymorphisms (SNPs) suggested that the wild lotus had the highest variant number (7 191 010). Population structure and genome diversity analysis indicated that the American wild lotus demonstrated a distant genetic relationship with the Asian lotus. Furthermore, the seed and rhizome lotus groups had not originated from a single source but rather had a more complex multisource origin. Besides that, the seed lotus showed higher genetic diversity, which might have been due to the gene flow from the flower lotus to seed lotus by artificial crossing, and the rhizome lotus showed a much lower genetic diversity than the other groups. The present study provides SNP markers for lotus genomic diversity analysis, which will be useful for guiding lotus breeding.

High expression of SARS-CoV-2 entry factors in human conjunctival goblet cells.

The angiotensin-converting enzyme 2 (ACE2) receptor has been proved for SARS-CoV-2 cell entry after auxiliary cellular protease priming by transmembrane protease serine 2 (TMPRSS2), but the co-effect of this molecular mechanism was unknown. Here, single-cell sequencing was performed with human conjunctiva and the results have shown that ACE2 and TMPRSS2 were highly co-expressed in the goblet cells with genes involved in immunity process. This identification of conjunctival cell types which are permissive to virus entry would help to understand the process by which SARS-CoV-2 infection was established. These finding might be suggestive for COVID-19 control and protection.

TFBSbank: a platform to dissect the big data of protein-DNA interaction in human and model species.

Genome-wide transcription factors (TFs) binding data has been extensively generated in the past few years, which poses a great challenge to data interpretation. Therefore, comprehensive and dedicated functional annotation databases for TF-DNA interaction are in great demands to manage, explore and utilize those invaluable data resources. Here, we constructed a platform 'TFBSbank' which houses the annotation of 1870 chromatin immunoprecipitation (ChIP) datasets of 585 TFs in five species (human, mouse, fly, worm and yeast). There are mainly five functional modules in TFBSbank aimed at characterizing ChIP peaks, identifying putative targets, predicting TF responsive enhancers, revealing potential cofactors/collaborators and discovering enriched TF motifs. TFBSbank has two distinctive features compared to the existing databases. Firstly, we provided putative cofactors/collaborators analysis (for Drosophila melanogaster), as they are crucial for the in vivo functions of TFs. Additionally, this database predicted the enrichment of both known and de novo motifs based on ChIP data. TFBSbank is freely accessible at http://tfbsbank.co.uk.

Virus Infection of Plants Alters Pollinator Preference: A Payback for Susceptible Hosts?

Plant volatiles play important roles in attraction of certain pollinators and in host location by herbivorous insects. Virus infection induces changes in plant volatile emission profiles, and this can make plants more attractive to insect herbivores, such as aphids, that act as viral vectors. However, it is unknown if virus-induced alterations in volatile production affect plant-pollinator interactions. We found that volatiles emitted by cucumber mosaic virus (CMV)-infected tomato (Solanum lycopersicum) and Arabidopsis thaliana plants altered the foraging behaviour of bumblebees (Bombus terrestris). Virus-induced quantitative and qualitative changes in blends of volatile organic compounds emitted by tomato plants were identified by gas chromatography-coupled mass spectrometry. Experiments with a CMV mutant unable to express the 2b RNA silencing suppressor protein and with Arabidopsis silencing mutants implicate microRNAs in regulating emission of pollinator-perceivable volatiles. In tomato, CMV infection made plants emit volatiles attractive to bumblebees. Bumblebees pollinate tomato by 'buzzing' (sonicating) the flowers, which releases pollen and enhances self-fertilization and seed production as well as pollen export. Without buzz-pollination, CMV infection decreased seed yield, but when flowers of mock-inoculated and CMV-infected plants were buzz-pollinated, the increased seed yield for CMV-infected plants was similar to that for mock-inoculated plants. Increased pollinator preference can potentially increase plant reproductive success in two ways: i) as female parents, by increasing the probability that ovules are fertilized; ii) as male parents, by increasing pollen export. Mathematical modeling suggested that over a wide range of conditions in the wild, these increases to the number of offspring of infected susceptible plants resulting from increased pollinator preference could outweigh underlying strong selection pressures favoring pathogen resistance, allowing genes for disease susceptibility to persist in plant populations. We speculate that enhanced pollinator service for infected individuals in wild plant populations might provide mutual benefits to the virus and its susceptible hosts.

Transcriptomic profiles of striped snakehead fish cells (SSN-1) infected with red-spotted grouper nervous necrosis virus (RGNNV) with an emphasis on apoptosis pathway.

Nervous necrosis virus (NNV), the causative agent of viral nervous necrosis (VNN) disease, has caused mass mortality of cultured marine and freshwater fish worldwide, resulting in enormous economic losses in the aquaculture industry. However, the molecular mechanisms underlying the pathogenicity of NNV are still poorly understood. In this study, the transcriptomic profiles of striped snakehead fish (Channa striatus) cells (SSN-1) infected with red-spotted grouper NNV (RGNNV) were investigated using deep RNA sequencing technique. From 254,955,234 raw reads, a total of 253,338,544 clean reads were obtained and they were assembled into 93,372 unigenes. Differentially expressed genes (DEGs) were identified from RGNNV-infected or mock-infected SSN-1 cells, including 1184 up-regulated and 1456 down-regulated genes at 3 h (h) post of infection (poi), and 1138 up-regulated and 2073 down-regulated genes at 24 h poi, respectively. These DEGs were involved in many pathways related to viral pathogenesis, including retinoic acid-inducible gene I (RIG-I) like receptors pathway, apoptosis pathway, oxidative phosphorylation, PI3K-Akt signaling pathway, and MAPK signaling pathway. Subsequent analysis focusing on the apoptosis pathway showed that the expression of Endonuclease G (EndoG) was up-regulated upon RGNNV infection at both 3 and 24 h poi. Therefore, EndoG gene was cloned and its function was further characterized. The results showed that over-expression of EndoG could also induce cellular apoptosis in SSN-1 cells, indicating that RGNNV infection might induce apoptosis of SSN-1 cells via EndoG-associated mitochondrial pathway. These results will shed a new light on the pathogenesis of NNV.

Cut&tag: a powerful epigenetic tool for chromatin profiling.

Analysis of transcription factors and chromatin modifications at the genome-wide level provides insights into gene regulatory processes, such as transcription, cell differentiation and cellular response. Chromatin immunoprecipitation is the most popular and powerful approach for mapping chromatin, and other enzyme-tethering techniques have recently become available for living cells. Among these, Cleavage Under Targets and Tagmentation (CUT&Tag) is a relatively novel chromatin profiling method that has rapidly gained popularity in the field of epigenetics since 2019. It has also been widely adapted to map chromatin modifications and TFs in different species, illustrating the association of these chromatin epitopes with various physiological and pathological processes. Scalable single-cell CUT&Tag can be combined with distinct platforms to distinguish cellular identity, epigenetic features and even spatial chromatin profiling. In addition, CUT&Tag has been developed as a strategy for joint profiling of the epigenome, transcriptome or proteome on the same sample. In this review, we will mainly consolidate the applications of CUT&Tag and its derivatives on different platforms, give a detailed explanation of the pros and cons of this technique as well as the potential development trends and applications in the future.

Nicotine-induced transcriptional changes and mitochondrial dysfunction in the ventral tegmental area revealed by single-nucleus transcriptomics.

Nicotine is widely recognized as the primary contributor to tobacco dependence. Previous studies have indicated that molecular and behavioral responses to nicotine are primarily mediated by ventral tegmental area (VTA) neurons, and accumulating evidence suggests that glia play prominent roles in nicotine addiction. However, VTA neurons and glia have yet to be characterized at the transcriptional level during the progression of nicotine self-administration. Here, a male mouse model of nicotine self-administration was established and the timing of three critical phases (pre-addiction, addicting, and post-addiction phase) was characterized. Single-nucleus RNA sequencing (snRNA-seq) in the VTA at each phase was performed to comprehensively classify specific cell subtypes. Adaptive changes occurred during the addicting and post-addiction phases, with the addicting phase displaying highly dynamic neuroplasticity that profoundly impacted the transcription in each cell subtype. Furthermore, significant transcriptional changes in energy metabolism-related genes were observed, accompanied by notable structural alterations in neuronal mitochondria during the progression of nicotine self-administration. The results provide insights into mechanisms underlying the progression of nicotine addiction, serving as important resource for identifying potential molecular targets for nicotine cessation.

Maize splicing-mediated mRNA surveillance impeded by sugarcane mosaic virus-coded pathogenic protein NIa-Pro.

The eukaryotic mRNA surveillance pathway, a pivotal guardian of mRNA fidelity, stands at the nexus of diverse biological processes, including antiviral immunity. Despite the recognized function of splicing factors on mRNA fate, the intricate interplay shaping the mRNA surveillance pathway remains elusive. We illustrate that the conserved splicing factor U2 snRNP auxiliary factor large subunit B (U2AF65B) modulates splicing of mRNA surveillance complex, contributing to transcriptomic homeostasis in maize. The functionality of the mRNA surveillance pathway requires ZmU2AF65B-mediated normal splicing of upstream frameshift 3 (ZmUPF3) pre-mRNA, encoding a core factor in this pathway. Intriguingly, sugarcane mosaic virus (SCMV)-coded nuclear inclusion protein a protease (NIa-Pro) hinders the splicing function of ZmU2AF65B. Furthermore, NIa-Pro disrupts ZmU2AF65B binding to ZmUPF3 pre-mRNA, leading to dysregulated splicing of ZmUPF3 transcripts and, consequently, impairing mRNA surveillance, thus facilitating viral infection. Together, this study establishes that splicing governs the mRNA surveillance pathway and identifies a pathogenic protein capable of disrupting this regulation to compromise RNA immunity.

The telomere-to-telomere (T2T) genome of Peucedanum praeruptorum Dunn provides insights into the genome evolution and coumarin biosynthesis.

BACKGROUND: Traditional Chinese medicine has used Peucedanum praeruptorum Dunn (Apiaceae) for a long time. Various coumarins, including the significant constituents praeruptorin (A-E), are the active constituents in the dried roots of P. praeruptorum. Previous transcriptomic and metabolomic studies have attempted to elucidate the distribution and biosynthetic network of these medicinal-valuable compounds. However, the lack of a high-quality reference genome impedes an in-depth understanding of genetic traits and thus the development of better breeding strategies. RESULTS: A telomere-to-telomere (T2T) genome was assembled for P. praeruptorum by combining PacBio HiFi, ONT ultra-long, and Hi-C data. The final genome assembly was approximately 1.798 Gb, assigned to 11 chromosomes with genome completeness >98%. Comparative genomic analysis suggested that P. praeruptorum experienced 2 whole-genome duplication events. By the transcriptomic and metabolomic analysis of the coumarin metabolic pathway, we presented coumarins' spatial and temporal distribution and the expression patterns of critical genes for its biosynthesis. Notably, the COSY and cytochrome P450 genes showed tandem duplications on several chromosomes, which may be responsible for the high accumulation of coumarins. CONCLUSIONS: A T2T genome for P. praeruptorum was obtained, providing molecular insights into the chromosomal distribution of the coumarin biosynthetic genes. This high-quality genome is an essential resource for designing engineering strategies for improving the production of these valuable compounds.

Biased pollen transfer by bumblebees favors the paternity of virus-infected plants in cross-pollination.

We used a green fluorescent protein marker gene for paternity analysis to determine if virus infection affected male reproductive success of tomato in bumblebee-mediated cross-pollination under glasshouse conditions. We found that bumblebees that visited flowers of infected plants showed a strong preference to subsequently visit flowers of non-infected plants. The behavior of the bumblebees to move toward non-infected plants after pollinating virus-infected plants appears to explain the paternity data, which demonstrate a statistically significant ∼10-fold bias for fertilization of non-infected plants with pollen from infected parents. Thus, in the presence of bumblebee pollinators, CMV-infected plants exhibit enhanced male reproductive success.

N 6-methyladenosine RNA methylation: From regulatory mechanisms to potential clinical applications.

Epitranscriptomics has emerged as another level of epigenetic regulation similar to DNA and histone modifications. N 6-methyladenosine (m6A) is one of the most prevalent and abundant posttranscriptional modifications, widely distributed in many biological species. The level of N 6-methyladenosine RNA methylation is dynamically and reversibly regulated by distinct effectors including methyltransferases, demethylases, histone modification and metabolites. In addition, N 6-methyladenosine RNA methylation is involved in multiple RNA metabolism pathways, such as splicing, localization, translation efficiency, stability and degradation, ultimately affecting various pathological processes, especially the oncogenic and tumor-suppressing activities. Recent studies also reveal that N 6-methyladenosine modification exerts the function in immune cells and tumor immunity. In this review, we mainly focus on the regulatory mechanisms of N 6-methyladenosine RNA methylation, the techniques for detecting N 6-methyladenosine methylation, the role of N 6-methyladenosine modification in cancer and other diseases, and the potential clinical applications.

A chromosomal level genome sequence for Quasipaa spinosa (Dicroglossidae) reveals chromosomal evolution and population diversity.

Quasipaa spinosa is an Asian commercial Dicroglossidae species noted for its spiny chest found in adult males. Here, we report the first chromosomal level Q. spinosa genome employing PacBio long read sequencing and high-resolution chromosome conformation capture (Hi-C) technology. The total length of the final assembled genome was 2,839,292,578 bp, with contig N50 of 3.79 Mb and scaffold N50 of 327.44 Mb. Approximately 99.30% of the length of the assembled genome sequences were anchored to 13 chromosomes with the assistance of Hi-C reads. A total of 26,173 protein-coding genes were predicted, and 95.98% of the genes were functionally annotated. The annotated genes covered a total of 92.10% of the complete vertebrate core gene set according to the BUSCO pipeline evaluation. Approximately 41 million years ago, Q. spinosa began to diverge from its dicroglossid sister taxon Nanorana parkeri. The Q. spinosa genome revealed obvious chromosomal fissions compared with Xenopus tropicalis, which probably represented a specific chromosome evolutionary history within frogs. Population analysis showed that Chinese Q. spinosa could be divided into eastern and western genetic clusters, with the western population showing higher diversity than the eastern population. The effective population size of Q. spinosa showed a continuously decreasing trend from one million years ago to 10,000 years ago. In summary, this study sheds light on Q. spinosa evolution and population differentiation, providing a valuable genomic resource for further biological and genetic studies on this species, and other closely related frog taxa.

Genome-wide characterization on MT family and their expression in response to environmental cues in upland cotton (Gossypium hirsutum L.).

Metallothioneins (MTs) are believed as key metal chelators and scavengers of reactive oxygen species (ROS), which are involved in tolerance and de-toxicity to multiple environmental stresses in plants. The MT gene family was characterized from upland cotton (Gossypium hirsutum L.), compared with its putative genome donors G. arboretum and raimondii. Subsequently, gene functions were predicted by promoter analysis. Moreover, gene expressions subjecting to exogenous stimuli, as well as in terms of developments, were studied. The main findings were shown as follows: 1) 19 GhMTs were identified from G. hirsutum, and the family completely included all four sub-types, namely p1, p2, p3, and pec. Sub-type p2 GhMTs were most conservative in protein motif compositions, gene structures, phylogenic relationships, and group numbers, while p3 GhMTs demonstrated much more diversiform and distant genetic relationships. 2) The GhMT family experienced apparent gene expansion, and the members from the D sub-genome were subjected to stronger environmental selection. 3) GhMTs played differential and overlapped roles in response to environmental cues. 4) GhMT6, GhMT8, and GhMT14 were involved in both vegetative and reproductive developments. These findings must provide valuable insights into understanding the plant MT gene family and novel gene resources for cotton breeding for environmental stresses, phytoremediation, and beyond.

Chromosome-level reference genome of tetraploid Isoetes sinensis provides insights into evolution and adaption of lycophytes.

BACKGROUND: The Lycophyta species are the extant taxa most similar to early vascular plants that were once abundant on Earth. However, their distribution has greatly diminished. So far, the absence of chromosome-level assembled lycophyte genomes has hindered our understanding of evolution and environmental adaption of lycophytes. FINDINGS: We present the reference genome of the tetraploid aquatic quillwort, Isoetes sinensis, a lycophyte. This genome represents the first chromosome-level assembled genome of a tetraploid seed-free plant. Comparison of genomes between I. sinensis and Isoetestaiwanensis revealed conserved and different genomic features between diploid and polyploid lycophytes. Comparison of the I. sinensis genome with those of other species representing the evolutionary lineages of green plants revealed the inherited genetic tools for transcriptional regulation and most phytohormones in I. sinensis. The presence and absence of key genes related to development and stress responses provide insights into environmental adaption of lycophytes. CONCLUSIONS: The high-quality reference genome and genomic analysis presented in this study are crucial for future genetic and environmental studies of not only I. sinensis but also other lycophytes.

Genomic basis of the giga-chromosomes and giga-genome of tree peony Paeonia ostii.

Tree peony (Paeonia ostii) is an economically important ornamental plant native to China. It is also notable for its seed oil, which is abundant in unsaturated fatty acids such as α-linolenic acid (ALA). Here, we report chromosome-level genome assembly (12.28 Gb) of P. ostii. In contrast to monocots with giant genomes, tree peony does not appear to have undergone lineage-specific whole-genome duplication. Instead, explosive LTR expansion in the intergenic regions within a short period (~ two million years) may have contributed to the formation of its giga-genome. In addition, expansion of five types of histone encoding genes may have helped maintain the giga-chromosomes. Further, we conduct genome-wide association studies (GWAS) on 448 accessions and show expansion and high expression of several genes in the key nodes of fatty acid biosynthetic pathway, including SAD, FAD2 and FAD3, may function in high level of ALAs synthesis in tree peony seeds. Moreover, by comparing with cultivated tree peony (P. suffruticosa), we show that ectopic expression of class A gene AP1 and reduced expression of class C gene AG may contribute to the formation of petaloid stamens. Genomic resources reported in this study will be valuable for studying chromosome/genome evolution and tree peony breeding.

A CpG Methylation Signature as a Potential Marker for Early Diagnosis of Hepatocellular Carcinoma From HBV-Related Liver Disease Using Multiplex Bisulfite Sequencing.

BACKGROUND: Aberrant methylation of CpG sites served as an epigenetic marker for building diagnostic, prognostic, and recurrence models for hepatocellular carcinoma (HCC). METHODS: Using Illumina 450K and EPIC Beadchip, we identified 34 CpG sites in peripheral blood mononuclear cell (PBMC) DNA that were differentially methylated in early HCC versus HBV-related liver diseases (HBVLD). We employed multiplex bisulfite sequencing (MBS) based on next-generation sequencing (NGS) to measure methylation of 34 CpG sites in PBMC DNA from 654 patients that were divided into a training set (n = 442) and a test set (n = 212). Using the training set, we selected and built a six-CpG-scorer (namely, cg14171514, cg07721852, cg05166871, cg18087306, cg05213896, and cg18772205), applying least absolute shrinkage and selection operator (LASSO) regression. We performed multivariable analyses of four candidate risk predictors (namely, six-CpG-scorer, age, sex, and AFP level), using 20 times imputation of missing data, non-linearly transformed, and backwards feature selection with logistic regression. The final model's regression coefficients were calculated according to "Rubin's Rules". The diagnostic accuracy of the model was internally validated with a 10,000 bootstrap validation dataset and then applied to the test set for validation. RESULTS: The area under the receiver operating characteristic curve (AUROC) of the model was 0.81 (95% CI, 0.77-0.85) and it showed good calibration and decision curve analysis. Using enhanced bootstrap validation, adjusted C-statistics and adjusted Brier score were 0.809 and 0.199, respectively. The model also showed an AUROC value of 0.84 (95% CI 0.79-0.88) of diagnosis for early HCC in the test set. CONCLUSIONS: Our model based on the six-CpG-scorer was a reliable diagnosis tool for early HCC from HBVLD. The usage of the MBS method can realize large-scale detection of CpG sites in clinical diagnosis of early HCC and benefit the majority of patients.

Transcriptional landscape of rice roots at the single-cell resolution.

There are two main types of root systems in flowering plants, namely taproot systems of dicots and fibrous root systems found in monocots. Despite this fundamental split, our current knowledge of cellular and molecular mechanism driving root development is mainly based on studies of the dicot model Arabidopsis. However, the world major crops are monocots and little is known about the transcriptional programs underlying cell-type specification in this clade. Here, we report the transcriptomes of more than 20 000 single cells derived from root tips of two agronomically important rice cultivars. Using combined computational and experimental analyses we were able to robustly identify most of the major cell types and define novel cell-type-specific marker genes for both cultivars. Importantly, we found divergent cell types associated with specific regulatory programs, including phytohormone biosynthesis, signaling, and response, which were well conserved between the two rice cultivars. In addition, we detected substantial differences between the cell-type transcript profiles of Arabidopsis and rice. These species-specific features emphasize the importance of analyzing tissues across diverse model species, including rice. Taken together, our study provides insight into the transcriptomic landscape of major cell types of rice root tip at single-cell resolution and opens new avenues to study cell-type specification, function, and evolution in plants.

Whole genome sequencing of silver carp (Hypophthalmichthys molitrix) and bighead carp (Hypophthalmichthys nobilis) provide novel insights into their evolution and speciation.

The edible silver carp (Hypophthalmichthys molitrix) and bighead carp (H. nobilis), which are two of the "Four Domesticated Fish" of China, are cultivated intensively worldwide. Here, we constructed 837- and 845-Mb draft genome assemblies for the silver carp and the bighead carp, respectively, including 24,571 and 24,229 annotated protein-coding genes. Genetic maps, anchoring 71.7% and 83.8% of all scaffolds, were obtained for the silver and bighead carp, respectively. Phylogenetic analysis showed that the bighead carp formed a clade with the silver carp, with an estimated divergence time of 3.6 million years ago; the time of divergence between the silver carp and zebrafish was 50.7 million years ago. An East Asian cyprinid genome-specific chromosome fusion took place ~9.2 million years after this clade diverged from the clade containing the common carp and Sinocyclocheilus. KEGG and GO analyses indicated that the expanded gene families in the silver and bighead carp were associated with diseases, the immune system and environmental adaptations. Genomic regions differentiating the silver and bighead carp populations were detected based on the whole-genome sequences of 42 individuals. Genes associated with the divergent regions were associated with reproductive system development and the development of primary female sexual characteristics. Thus, our results provided a novel systematic genomic analysis of the East Asian cyprinids, as well as the evolution and speciation of the silver carp and bighead carp.