Table_1_Application of third-generation sequencing to herbal genomics.docx

There is a long history of traditional medicine use. However, little genetic information is available for the plants used in traditional medicine, which limits the exploitation of these natural resources. Third-generation sequencing (TGS) techniques have made it possible to gather invaluable genetic information and develop herbal genomics. In this review, we introduce two main TGS techniques, PacBio SMRT technology and Oxford Nanopore technology, and compare the two techniques against Illumina, the predominant next-generation sequencing technique. In addition, we summarize the nuclear and organelle genome assemblies of commonly used medicinal plants, choose several examples from genomics, transcriptomics, and molecular identification studies to dissect the specific processes and summarize the advantages and disadvantages of the two TGS techniques when applied to medicinal organisms. Finally, we describe how we expect that TGS techniques will be widely utilized to assemble telomere-to-telomere (T2T) genomes and in epigenomics research involving medicinal plants.</p

DataSheet1_TaqMan Probe-Based Quantitative Real-Time PCR to Detect Panax notoginseng in Traditional Chinese Patent Medicines.docx

Background: There has been global concern about the safety and accuracy of traditional Chinese patent medicines (TCPMs). Panax notoginseng, also known as sanqi, is an important constituent of TCPMs. However, identifying the species contained in TCPMs is challenging due to the presence of multiple ingredients and the use of various preparation processes.Objective: To detect P. notoginseng in TCPMs.Methods: A TaqMan probe-based qPCR assay was constructed and validated with DNA extracted from P. notoginseng and adulterants. In total, 75 samples derived from 25 batches of TCPMs were tested using the constructed qPCR method.Results: A TaqMan probe-based qPCR assay targeting P. notoginseng was established. The constructed qPCR assay could specifically discriminate P. notoginseng from Panax ginseng, Panax quinquefolium and Curcuma aromatica Salisb. cv. Wenyujin. The sensitivity study showed that the detectable DNA template concentration of P. notoginseng for this qPCR assay was 0.001 ng/μl. All 75 samples from TCPMs were confirmed to contain P. notoginseng by the qPCR assay.Conclusions: The qPCR method can accurately identify P. notoginseng in TCPMs and is promising as a powerful tool for quality control and market regulation.</p

Data_Sheet_1_Analysis of Whole-Genome facilitates rapid and precise identification of fungal species.docx

Fungal identification is a cornerstone of fungal research, yet traditional molecular methods struggle with rapid and accurate onsite identification, especially for closely related species. To tackle this challenge, we introduce a universal identification method called Analysis of whole GEnome (AGE). AGE includes two key steps: bioinformatics analysis and experimental practice. Bioinformatics analysis screens candidate target sequences named Targets within the genome of the fungal species and determines specific Targets by comparing them with the genomes of other species. Then, experimental practice using sequencing or non-sequencing technologies would confirm the results of bioinformatics analysis. Accordingly, AGE obtained more than 1,000,000 qualified Targets for each of the 13 fungal species within the phyla Ascomycota and Basidiomycota. Next, the sequencing and genome editing system validated the ultra-specific performance of the specific Targets; especially noteworthy is the first-time demonstration of the identification potential of sequences from unannotated genomic regions. Furthermore, by combining rapid isothermal amplification and phosphorothioate-modified primers with the option of an instrument-free visual fluorescence method, AGE can achieve qualitative species identification within 30 min using a single-tube test. More importantly, AGE holds significant potential for identifying closely related species and differentiating traditional Chinese medicines from their adulterants, especially in the precise detection of contaminants. In summary, AGE opens the door for the development of whole-genome-based fungal species identification while also providing guidance for its application in plant and animal kingdoms.</p

Additional file 1: of A novel mutation of MSX1 in oligodontia inhibits odontogenesis of dental pulp stem cells via the ERK pathway

Figure S1. ERK activity is not required for MSX1 expression. (A) Immunolocalization of flag-tagged MSX1 protein in U0126-treated DPSCs. Wild-type MSX1 located exclusively in the nucleus, while mutant MSX1 was distributed over the entire cytoplasm. (B) U0126-treated DPSCs showed comparable levels of MSX1 expression as control. WT wild-type MSX1 transfection, MT mutant MSX1 transfection, Control no transfection, U0126 U0126 added. (PDF 2680 kb

Data_Sheet_1_Identification of closely related species in Aspergillus through Analysis of Whole-Genome.docx

The challenge of discriminating closely related species persists, notably within clinical diagnostic laboratories for invasive aspergillosis (IA)-related species and food contamination microorganisms with toxin-producing potential. We employed Analysis of the whole-GEnome (AGE) to address the challenges of closely related species within the genus Aspergillus and developed a rapid detection method. First, reliable whole genome data for 77 Aspergillus species were downloaded from the database, and through bioinformatic analysis, specific targets for each species were identified. Subsequently, sequencing was employed to validate these specific targets. Additionally, we developed an on-site detection method targeting a specific target using a genome editing system. Our results indicate that AGE has successfully achieved reliable identification of all IA-related species (Aspergillus fumigatus, Aspergillus niger, Aspergillus nidulans, Aspergillus flavus, and Aspergillus terreus) and three well-known species (A. flavus, Aspergillus parasiticus, and Aspergillus oryzae) within the Aspergillus section. Flavi and AGE have provided species-level-specific targets for 77 species within the genus Aspergillus. Based on these reference targets, the sequencing results targeting specific targets substantiate the efficacy of distinguishing the focal species from its closely related species. Notably, the amalgamation of room-temperature amplification and genome editing techniques demonstrates the capacity for rapid and accurate identification of genomic DNA samples at a concentration as low as 0.1 ng/μl within a concise 30-min timeframe. Importantly, this methodology circumvents the reliance on large specialized instrumentation by presenting a singular tube operational modality and allowing for visualized result assessment. These advancements aptly meet the exigencies of on-site detection requirements for the specified species, facilitating prompt diagnosis and food quality monitoring. Moreover, as an identification method based on species-specific genomic sequences, AGE shows promising potential as an effective tool for epidemiological research and species classification.</p

Additional file 1 of A novel mutation of SATB2 inhibits odontogenesis of human dental pulp stem cells through Wnt/β-catenin signaling pathway

Additional file 1. Supplementary Figure 1. Living/dead viability assay of cells after transfection of wild-type and mutant SATB2. Cells stained with calcein AM in green manifested living hDPSCS while cells stained with ethidium homodimer-1 (EthD-1) in red were dead. Both wild-type and mutant SATB2 decreased cell viability compared with control, but no significant difference was found between wild-type and mutant. **P < 0.01, NS non-significant. Data are expressed as the means+SD. Each experiment was repeated three times with n ≥ 3 samples per group. Supplementary Table 1. Primer list for quantitative real-time PCR