Influence of storage solution, temperature, assay time and concentration on RT-qPCR nucleic acid detection for SARS-CoV-2 detection of SARS-CoV-2 by the RT-qPCR.

Real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is an important method for the early diagnosis of coronavirus disease 2019 (COVID-19). This study investigated the effects of storage solution, temperature and detection time on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection by RT-qPCR. Various concentrations of SARS-CoV-2 were added to inactive and non-inactive storage solution and the viral suspensions were stored at various temperatures (room temperature, 4, -20 and -80 °C). Then, at five different detection time points, the Ct values were determined by RT-qPCR. Active and inactive storage solutions and storage temperature have a great impact on the detection of N gene of SARS-CoV-2 at different concentration corridors but have little impact on the ORF gene. The storage time has a greater impact on the N gene and ORF gene at high concentrations but has no effect on the two genes at low concentrations. In conclusion, storage temperature, storage time and storage status (inactivated, non-inactivated) have no effect on the nucleic acid detection of SARS-CoV-2 at the same concentration. For different concentrations of SARS-CoV-2, the detection of N gene is mainly affected.

Chromosome-scale genome assembly of sweet tea (Lithocarpus polystachyus Rehder).

Lithocarpus, with >320 species, is the second largest genus of Fagaceae. However, the lack of a reference genome limits the molecular biology and functional study of Lithocarpus species. Here, we report the chromosome-scale genome assembly of sweet tea (Lithocarpus polystachyus Rehder), the first Lithocarpus species to be sequenced to date. Sweet tea has a 952-Mb genome, with a 21.4-Mb contig N50 value and 98.6% complete BUSCO score. In addition, the per-base consensus accuracy and completeness of the genome were estimated at 60.6 and 81.4, respectively. Genome annotation predicted 37,396 protein-coding genes, with repetitive sequences accounting for 64.2% of the genome. The genome did not undergo whole-genome duplication after the gamma (γ) hexaploidy event. Phylogenetic analysis showed that sweet tea diverged from the genus Quercus approximately at 59 million years ago. The high-quality genome assembly and gene annotation resources enrich the genomics of sweet tea, and will facilitate functional genomic studies in sweet tea and other Fagaceae species.

Molecular epidemiological characteristics of SARS-CoV-2 in imported cases from 2021 to 2022 in Zhejiang Province, China.

Backgrounds: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a global threat since 2020. The emergence of the Omicron variant in 2021, which replaced Delta as the dominant variant of concern, has had a significant adverse impact on the global economy and public health. During this period, Zhejiang Province implemented dynamic zeroing and focused on preventing imported cases. This study aimed to gain clear insight into the characteristics of imported COVID-19 cases in Zhejiang Province. Methods: We conducted a systematic molecular epidemiological analysis of 146 imported cases between July 2021 and November 2022 in Zhejiang Province. Virus samples with cycle threshold (Ct) value less than 32 were performed next generation sequencing. Basing the whole genome sequence obtained after quality control and assembly of reads, the whole genome variation map and phylogenetic tree were constructed and further analyzed. Results: Our study identified critical months and populations for surveillance, profiled the variation of various lineages, determined the evolutionary relationships among various lineages of SARS-CoV-2, and compared the results in Zhejiang with those obtained worldwide during this period. Conclusion: The continuous molecular epidemiological surveillance of imported cases of COVID-19 in Zhejiang Province during 2021 to 2022 is consistent with the global epidemic trend.

Epidemiology of hand, foot and mouth disease and genomic surveillance of coxsackievirus A10 circulating in Zhejiang Province, China during 2017 to 2022.

BACKGROUND: Coxsackievirus A10 (CA10) is one of the etiological agents associated with hand, foot and mouth disease (HFMD). OBJECTIVES: We aimed to perform a retrospective analysis of the molecular epidemiological characteristics and genetic features of HFMD associated with CA10 infections in Zhejiang Province from 2017 to 2022. STUDY DESIGN: Epidemiologic features were summarized. Throat swab specimens were collected and tested. The VP1 regions were sequenced for genotyping. CA10 positive samples were isolated. Whole genomes of CA10 isolations were sequenced. Nucleotide and amino acid changes were characterized. Phylogenetic trees were constructed. RESULTS: The number of HFMD cases fluctuated from 2017 to 2022. Children aged below 3 years accounted for the majority (66.29%) and boys were more frequently affected than girls. Cases peaked in June. The positivity rate of HEV was 62.69%. A total of 90 strains of CA10 were isolated and 53 genomes were obtained. All CA10 in this study could be assigned to two genogroups, C (C2) and F (F1 and F3). CONCLUSION: The clinical manifestations of HFMD associated with HEV are complex and diverse. CA10 infection may be emerging as a new and major cause of HFMD because an upward trend was observed in the proportion of CA10 cases after the use of EV71 vaccines. Different genogroups of CA10 had different geographic distribution patterns. Surveillance should be strengthened and further comprehensive studies should be continued to provide a scientific basis for HFMD prevention and control.

Genome-Wide Identification and Expression Analysis of Calmodulin-Like Gene Family in Paspalums vaginatium Revealed Their Role in Response to Salt and Cold Stress.

The calmodulin-like (CML) family is an important calcium (Ca2+) sensor in plants and plays a pivotal role in the response to abiotic and biotic stresses. As one of the most salt-tolerant grass species, Paspalums vaginatum is resistant to multiple abiotic stresses, such as salt, cold, and drought. However, investigations of PvCML proteins in P. vaginatum have been limited. Based on the recently published P. vaginatum genome, we identified forty-nine PvCMLs and performed a comprehensive bioinformatics analysis of PvCMLs. The main results showed that the PvCMLs were unevenly distributed on all chromosomes and that the expansion of PvCMLs was shaped by tandem and segmental duplications. In addition, cis-acting element analysis, expression profiles, and qRT-PCR analysis revealed that PvCMLs were involved in the response to salt and cold stress. Most interestingly, we found evidence of a tandem gene cluster that independently evolved in P. vaginatum and may participate in cold resistance. In summary, our work provides important insight into how grass species are resistant to abiotic stresses such as salt and cold and could be the basis of further gene function research on CMLs in P. vaginatum.

Genomic vulnerability to climate change in Quercus acutissima, a dominant tree species in East Asian deciduous forests.

Understanding the evolutionary processes that shape the landscape of genetic variation and influence the response of species to future climate change is critical for biodiversity conservation. Here, we sampled 27 populations across the distribution range of a dominant forest tree, Quercus acutissima, in East Asia, and applied genome-wide analyses to track the evolutionary history and predict the fate of populations under future climate. We found two genetic groups (East and West) in Q. acutissima that diverged during Pliocene. We also found a heterogeneous landscape of genomic variation in this species, which may have been shaped by population demography and linked selections. Using genotype-environment association analyses, we identified climate-associated SNPs in a diverse set of genes and functional categories, indicating a model of polygenic adaptation in Q. acutissima. We further estimated three genetic offset metrics to quantify genomic vulnerability of this species to climate change due to the complex interplay between local adaptation and migration. We found that marginal populations are under higher risk of local extinction because of future climate change, and may not be able to track suitable habitats to maintain the gene-environment relationships observed under the current climate. We also detected higher reverse genetic offsets in northern China, indicating that genetic variation currently present in the whole range of Q. acutissima may not adapt to future climate conditions in this area. Overall, this study illustrates how evolutionary processes have shaped the landscape of genomic variation, and provides a comprehensive genome-wide view of climate maladaptation in Q. acutissima.

Phylogenomic analyses highlight innovation and introgression in the continental radiations of Fagaceae across the Northern Hemisphere.

Northern Hemisphere forests changed drastically in the early Eocene with the diversification of the oak family (Fagaceae). Cooling climates over the next 20 million years fostered the spread of temperate biomes that became increasingly dominated by oaks and their chestnut relatives. Here we use phylogenomic analyses of nuclear and plastid genomes to investigate the timing and pattern of major macroevolutionary events and ancient genome-wide signatures of hybridization across Fagaceae. Innovation related to seed dispersal is implicated in triggering waves of continental radiations beginning with the rapid diversification of major lineages and resulting in unparalleled transformation of forest dynamics within 15 million years following the K-Pg extinction. We detect introgression at multiple time scales, including ancient events predating the origination of genus-level diversity. As oak lineages moved into newly available temperate habitats in the early Miocene, secondary contact between previously isolated species occurred. This resulted in adaptive introgression, which may have further amplified the diversification of white oaks across Eurasia.

Globally Relaxed Selection and Local Adaptation in Boechera stricta.

The strength of selection varies among populations and across the genome, but the determinants of efficacy of selection remain unclear. In this study, we used whole-genome sequencing data from 467 Boechera stricta accessions to quantify the strength of selection and characterize the pattern of local adaptation. We found low genetic diversity on 0-fold degenerate sites and conserved non-coding sites, indicating functional constraints on these regions. The estimated distribution of fitness effects and the proportion of fixed substitutions suggest relaxed negative and positive selection in B. stricta. Among the four population groups, the NOR and WES groups have smaller effective population size (Ne), higher proportions of effectively neutral sites, and lower rates of adaptive evolution compared with UTA and COL groups, reflecting the effect of Ne on the efficacy of natural selection. We also found weaker selection on GC-biased sites compared with GC-conservative (unbiased) sites, suggested that GC-biased gene conversion has affected the strength of selection in B. stricta. We found mixed evidence for the role of the recombination rate on the efficacy of selection. The positive and negative selection was stronger in high-recombination regions compared with low-recombination regions in COL but not in other groups. By scanning the genome, we found different subsets of selected genes suggesting differential adaptation among B. stricta groups. These results show that differences in effective population size, nucleotide composition, and recombination rate are important determinants of the efficacy of selection. This study enriches our understanding of the roles of natural selection and local adaptation in shaping genomic variation.

Linked selection shapes the landscape of genomic variation in three oak species.

Natural selection shapes genome-wide patterns of diversity within species and divergence between species. However, quantifying the efficacy of selection and elucidating the relative importance of different types of selection in shaping genomic variation remain challenging. We sequenced whole genomes of 101 individuals of three closely related oak species to track the divergence history, and to dissect the impacts of selective sweeps and background selection on patterns of genomic variation. We estimated that the three species diverged around the late Neogene and experienced a bottleneck during the Pleistocene. We detected genomic regions with elevated relative differentiation ('FST -islands'). Population genetic inferences from the site frequency spectrum and ancestral recombination graph indicated that FST -islands were formed by selective sweeps. We also found extensive positive selection; the fixation of adaptive mutations and reduction neutral diversity around substitutions generated a signature of selective sweeps. Prevalent negative selection and background selection have reduced genetic diversity in both genic and intergenic regions, and contributed substantially to the baseline variation in genetic diversity. Our results demonstrate the importance of linked selection in shaping genomic variation, and illustrate how the extent and strength of different selection models vary across the genome.

Molecular epidemiologic characteristics of hemagglutinin from five waves of avian influenza A (H7N9) virus infection, from 2013 to 2017, in Zhejiang Province, China.

There have been five waves of influenza A (H7N9) epidemics in Zhejiang Province between 2013 and 2017. Although the epidemiological characteristics of the five waves have been reported, the molecular genetics aspects, including the phylogeny, evolution, and mutation of hemagglutinin (HA), have not been systematically investigated. A total of 154 H7N9 samples from Zhejiang Province were collected between 2013 and 2017 and sequenced using an Ion Torrent Personal Genome Machine. The starting dates of the waves were 16 March 2013, 1 July 2013, 1 July 2014, 1 July 2015, and 1 July 2016. Single-nucleotide polymorphisms (SNPs) and amino acid mutations were counted after the HA sequences were aligned. The evolution of H7N9 matched the temporal order of the five waves, among which wave 3 played an important role. The 55 SNPs and 14 amino acid mutations with high frequency identified among the five waves revealed the dynamic occurrence of mutation in the process of viral dissemination. Wave 3 contributed greatly to the subsequent epidemic of waves 4 and 5 of H7N9. Compared with wave 1, wave 5 was characterized by more mutations, including A143V and R148K, two mutations that have been reported to weaken the immune response. In addition, some amino acid mutations were observed in wave 5 that led to more lineages. It is necessary to strengthen the surveillance of subsequent H7N9 influenza outbreaks.

scDPN for High-throughput Single-cell CNV Detection to Uncover Clonal Evolution During HCC Recurrence.

Single-cell genomics provides substantial resources for dissecting cellular heterogeneity and cancer evolution. Unfortunately, classical DNA amplification-based methods have low throughput and introduce coverage bias during sample preamplification. We developed a single-cell DNA library preparation method without preamplification in nanolitre scale (scDPN) to address these issues. The method achieved a throughput of up to 1800 cells per run for copy number variation (CNV) detection. Also, our approach demonstrated a lower level of amplification bias and noise than the multiple displacement amplification (MDA) method and showed high sensitivity and accuracy for cell line and tumor tissue evaluation. We used this approach to profile the tumor clones in paired primary and relapsed tumor samples of hepatocellular carcinoma (HCC). We identified three clonal subpopulations with a multitude of aneuploid alterations across the genome. Furthermore, we observed that a minor clone of the primary tumor containing additional alterations in chromosomes 1q, 10q, and 14q developed into the dominant clone in the recurrent tumor, indicating clonal selection during recurrence in HCC. Overall, this approach provides a comprehensive and scalable solution to understand genome heterogeneity and evolution.

Small Intestinal Tuft Cell Activity Associates With Energy Metabolism in Diet-Induced Obesity.

Little is known about the involvement of type 2 immune response-promoting intestinal tuft cells in metabolic regulation. We here examined the temporal changes in small intestinal tuft cell number and activity in response to high-fat diet-induced obesity in mice and investigated the relation to whole-body energy metabolism and the immune phenotype of the small intestine and epididymal white adipose tissue. Intake of high fat diet resulted in a reduction in overall numbers of small intestinal epithelial and tuft cells and reduced expression of the intestinal type 2 tuft cell markers Il25 and Tslp. Amongst >1,700 diet-regulated transcripts in tuft cells, we observed an early association between body mass expansion and increased expression of the gene encoding the serine protease inhibitor neuroserpin. By contrast, tuft cell expression of genes encoding gamma aminobutyric acid (GABA)-receptors was coupled to Tslp and Il25 and reduced body mass gain. Combined, our results point to a possible role for small intestinal tuft cells in energy metabolism via coupled regulation of tuft cell type 2 markers and GABA signaling receptors, while being independent of type 2 immune cell involvement. These results pave the way for further studies into interventions that elicit anti-obesogenic circuits via small intestinal tuft cells.

Single-cell differential splicing analysis reveals high heterogeneity of liver tumor-infiltrating T cells.

Recent advances in single-cell RNA sequencing (scRNA-seq) have improved our understanding of the association between tumor-infiltrating lymphocyte (TILs) heterogeneity and cancer initiation and progression. However, studies investigating alternative splicing (AS) as an important regulatory factor of heterogeneity remain limited. Here, we developed a new computational tool, DESJ-detection, which accurately detects differentially expressed splicing junctions (DESJs) between cell groups at the single-cell level. We analyzed 5063 T cells of hepatocellular carcinoma (HCC) and identified 1176 DESJs across 11 T cell subtypes. Interestingly, DESJs were enriched in UTRs, and have putative effects on heterogeneity. Cell subtypes with a similar function closely clustered together at the AS level. Meanwhile, we identified a novel cell state, pre-activation with the isoform markers ARHGAP15-205. In summary, we present a comprehensive investigation of alternative splicing differences, which provided novel insights into T cell heterogeneity and can be applied to other full-length scRNA-seq datasets.

Single-cell RNA profiling links ncRNAs to spatiotemporal gene expression during C. elegans embryogenesis.

Recent studies show that non-coding RNAs (ncRNAs) can regulate the expression of protein-coding genes and play important roles in mammalian development. Previous studies have revealed that during C. elegans (Caenorhabditis elegans) embryo development, numerous genes in each cell are spatiotemporally regulated, causing the cell to differentiate into distinct cell types and tissues. We ask whether ncRNAs participate in the spatiotemporal regulation of genes in different types of cells and tissues during the embryogenesis of C. elegans. Here, by using marker-free full-length high-depth single-cell RNA sequencing (scRNA-seq) technique, we sequence the whole transcriptomes from 1031 embryonic cells of C. elegans and detect 20,431 protein-coding genes, including 22 cell-type-specific protein-coding markers, and 9843 ncRNAs including 11 cell-type-specific ncRNA markers. We induce a ncRNAs-based clustering strategy as a complementary strategy to the protein-coding gene-based clustering strategy for single-cell classification. We identify 94 ncRNAs that have never been reported to regulate gene expressions, are co-expressed with 1208 protein-coding genes in cell type specific and/or embryo time specific manners. Our findings suggest that these ncRNAs could potentially influence the spatiotemporal expression of the corresponding genes during the embryogenesis of C. elegans.