Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing

Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing.Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing.Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing.Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.

Variation of microbiological and small molecule metabolite profiles of Nuodeng ham during ripening by high-throughput sequencing and GC-TOF-MS

The internal microbial diversity and small molecular metabolites of Nuodeng ham in different processing years(the first,second and third year sample)were analyzed by high-throughput sequencing technology and gas chromatography-time of flight mass spectrography(GC-TOF-MS)to study the effects of microorganisms and small molecular metabolites on the quality of ham in different processing years.The results showed that the dominant bacteria phyla of Nuodeng ham in different processing years were Proteobacteria and Firmicutes,the dominant fungi phyla were Ascomycota and Basidiomycota,while Staphylococcus and Aspergillus were the dominant bacteria and fungi of Nuodeng ham,respectively.Totally,252 kinds of small molecular metabolites were identified from Nuodeng ham in different processing years,and 12 different metabolites were screened through multivariate statistical analysis.Further metabolic pathway analysis showed that 23 metabolic pathways were related to ham fermentation,of which 8 metabolic pathways had significant effects on ham fermentation(Impact>0.01,P<0.05).The content of L-proline,phenyllactic acid,L-lysine,carnosine,taurine,D-proline,betaine and creatine were significantly positively correlated with the relative abundance of Staphylococcus and Serratia,but negatively correlated with the relative abundance of Halomonas,Aspergillus and Yamadazyma.

Development and Characterization of Microsatellite Markers for Harpadon nehereus Based on High-Throughput Sequencing and Cross-Species Amplification in Three Myctophiformes Fishes

Harpadon nehereus is a widespread economical fish found in the coastal seas of China and has important ecological value in the marine ecosystem.H_(o)wever,its germplasm resources have been seriously degraded due to natural factors and anthropogenic activities.In this study,high-throughput sequencing was applied to search for microsatellite loci in H.nehereus transcriptome to provide references for its resource conservation and utilization.Polymorphic loci were developed by non-denaturing polyacrylamide gel electrophoresis,and their cross-species amplified ability was detected in three related species.A total of 5652 microsatellites were identified from 16974320 unigenes.Among the primer pairs designed for 100 SSRs for PCR amplification,80%were successfully amplified,and 26 loci were polymorphic with a high number of alleles from 3 to 11 each.The expected(H_(e))and observed(H_(o))heterozygosities were 0.355–0.885 and 0.375–0.958,respectively.Most of the loci were highly polymorphic(polymorphism information content:0.316–0.852;mean:0.713),and these markers can be applied in the population genetic diversity research of H.nehereus.H_(o)wever,the transferability of these primers was low,probably because of the close relation of the collected species.In follow-up work,simple sequence repeats will be excavated with genome-based technologies,and related species will be gathered to address the present inadequacies.

Comparison of rumen archaeal diversity in adult and elderly yaks(Bos grunniens)using 16S rRNA gene high-throughput sequencing

This study was conducted to investigate the phylogenetic diversity of archaea in the rumen of adult and elderly yaks. Six domesticated female yaks, 3 adult yaks （（5.3±0.6） years old）, and 3 elderly yaks （（10.7±0.6） years old）, were used for the rumen contents collection. Illumina MiSeq high-throughput sequencing technology was applied to examine the archaeal composition of rumen contents. A total of 92 901 high-quality archaeal sequences were analyzed, and these were assigned to 2 033 operational taxonomic units （OTUs）. Among these, 974 OTUs were unique to adult yaks while 846 OTUs were unique to elderly yaks; 213 OTUs were shared by both groups. At the phylum level, more than 99% of the obtained OTUs belonged to the Euryarchaeota phylum. At the genus level, the archaea could be divided into 7 archaeal genera. The 7 genera （i.e., Methanobrevibacter, Methanobacterium, Methanosphaera, Thermogymnomonas, Methanomicrobiu, Meth- animicrococcus and the unclassified genus） were shared by all yaks, and their total abundance accounted for 99% of the rumen archaea. The most abundant archaea in elderly and adult yaks were Methanobrevibacterand Thermogymnomonas, respectively. The abundance of Methanobacteria （class）, Methanobacteriales （order）, Methanobacteriaceae （family）, and Methanobrevibacter （genus） in elderly yaks was significantly higher than in adult yaks. In contrast, the abundance of Ther-mogymnomonas in elderly yaks was 34% lower than in adult yaks, though the difference was not statistically significant. The difference in abundance of other archaea was not significant between the two groups. These results suggested that the structure of archaea in the rumen of yaks changed with age. This is the first study to compare the phytogenetic differences of rumen archaeal structure and composition using the yak model.

High-throughput Sequencing Technology and Its Application

Gene sequencing is a great way to interpret life, and high-throughput sequencing technology is a revolutionary technological innovation in gene sequencing researches. This technology is characterized by low cost and high-throughput data. Currently, high-throughput sequencing technology has been widely applied in multi-level researches on genomics, transcriptomics and epigenomics. And it has fundamentally changed the way we approach problems in basic and translational researches and created many new possibilities. This paper presented a general description of high-throughput sequencing technology and a comprehensive review of its application with plain, concisely and precisely. In order to help researchers finish their work faster and better, promote science amateurs and understand it easier and better.

High-throughput sequencing analysis of the regulation of intestinal flora in giant pandas with indigestion using a probiotic agent LyPB

This study aimed to investigate the eff ect of LyPB on the intestinal microfl ora of giant pandas with indigestion,using high-throughput sequencing(HTS)technology.The species distribution and microfl oral density and diversity before and after administration of the LyPB probiotic agent were analyzed.LyPB evidently has the ability to adjust the fl oral imbalance in the panda’s intestine.To test the eff ects of LyPB on the microfl ora of the panda gut,fecal samples were taken from a healthy giant panda(Anan)without administration of LyPB and from a dyspeptic giant panda Yangyang before and after LyPB administration.Compared with the sample obtained from healthy Anan(anan-c)and that obtained from dyspeptic Yangyang before LyPB administration(yangyang1),the sample taken from Yangyang(yangyang2)after LyPB administration displayed a signifi cant increase in the operational taxonomic unit index.An increase in the Chao index indicated an increase in the microfl oral richness,while an increase in the Shannon index indicated an increase in microfl oral diversity.At phylum and genus levels,a signifi cant increase was observed in the density of probiotic bacteria of phylum fi rmicutes,genus Streptococcus,while a drastic reduction in the density of Escherichia coli/Escherichia coli Shigella/bacteria of genus Shigella was observed.Data obtained in this study shows that LyPB preparations successfully improve the microbial structure within the panda’s intestinal canal by signifi cantly increasing the eff ective microbial community and decreasing the number of pathogenic microbes.

Characterization of Bacterial Communities Associating with Larval Development of Yesso Scallop(Patinopecten yessoensisis Jay, 1857) by High-Throughput Sequencing

Bacterial community presumably plays an essential role in inhibiting pathogen colonization and maintaining the health of scallop larvae, but limiting data are available for Yesso scallop （Patinopecten yessoensisis Jay, 1857） larval development stages. The aim of this study was to characterize and compare the bacterial communities associating with Yesso scallop larval development at fertilized egg S l, trochophora S2, D-shaped larvae S3, umbo larvae S4, and juvenile scallop S5 stages by Illumina high-throughput sequencing. Genomic DNA was extracted from the larvae and their associating baetera, and a gene segment covering V3-V4 region of 16S rRNA gene was amplified and sequenced using an Illumina Miseq sequencer. Overall, 106760 qualified sequences with an average length of 449 bp were obtained. Sequences were compared with those retrieved from 16S rRNA gene databases, and 4 phyla, 7 classes, 15 orders, 21 families, 31 genera were identified. Proteobacteria was predominant phylum, accounting for more than 99%, at all 5 larval development stages. At genus level, Pseudomonas was dominant at stages S1 （80.60%）, S2 （87.77%） and S5 （68.71%）, followed by Photobacterium （17.06%） and Aeromonas （1.64%） at stage S1, Serratia （6.94%）, Stenotrophomonas （3.08%） and Acinetobacter （1.2%） at stage S2, Shewanella （25.95%） and Pseudoalteromonas （4.57%） at stage S5. Moreover, genus Pseudoal- teromonas became dominant at stages S3 （44.85%） and S4 （56.02%）, followed by Photobacterium （29.82%）, Pseudomonas （11.86%）, Aliivibrio （8.60%） and Shewanella （3.39%） at stage S3, Pseudomonas （18.16%）, Aliivibrio （14.29%）, Shewanella （4.11%）, Psychro- monas （4.04%） and Psychrobacter （1.81%） at stage S4. From the results, we concluded that the bacterial community changed sig- nificantly at different development stages of Yesso Scallop larvae.

High-throughput sequencing exclusively identified a novel Torque teno virus genotype in serum of a patient with fatal fever

Torque teno virus(TTV) has been found to be prevalent world-wide in healthy populations and in patients with various diseases, but its etiological role has not yet been determined. Using high-throughput unbiased sequencing to screen for viruses in the serum of a patient with persistent high fever who died of suspected viral infection and prolonged weakness, we identified the complete genome sequence of a TTV(isolate Hebei-1). The genome of TTV-Hebei-1 is 3649 bp in length, encoding four putative open reading frames, and it has a G+C content of 49%. Genomic comparison and a BLASTN search revealed that the assembled genome of TTV-Hebei-1 represented a novel isolate, with a genome sequence that was highly heterologous to the sequences of other reported TTV strains. A phylogenetic tree constructed using the complete genome sequence showed that TTV-Hebei-1 and an uncharacterized Taiwan Residents strain, TW53A37, constitute a new TTV genotype. The patient was strongly suspected of carrying a viral infection and died eventually without any other possible causes being apparent. No virus other than the novel TTV was identified in his serum sample. Although a direct causal link between the novel TTV genotype infection and the patient's disease could not be confirmed, the findings suggest that surveillance of this novel TTV genotype is necessary and that its role in disease deserves to be explored.

Profile and development of microsatellite primers for Acanthogobius ommaturus based on high-throughput sequencing technology

Acanthogobius ommaturus,a fish species of the Family Gobiidae,is a marine commercial fish perched on the bottom of seawater.In this study,Illumina high-throughput sequencing technology was applied to obtain the candidate microsatellite markers of A.ommaturus.A total of 4746 microsatellite-rich fragments were found,of which 4542 microsatellites are with primer fragments,containing 971 dinucleotide sequences,2643 trinucleotide sequences,569 tetranucleotide sequences,406 pentanucleotide sequences,and 212 hexanucleotide sequences.Based on the results of high-throughput sequencing,a total of 141 pairs of the microsatellite primers were designed and screened.And then 24 polymorphic primers were finally obtained by polyacrylamide gel electrophoresis.In total,271 alleles were detected in the 24 pairs of primers.The number of alleles for different primers ranged from 5 to 19.The average number of effective alleles(Na)was 11.292;the average observed heterozygosity(Ho)of the 24 pairs of primers was 0.665,the average expected heterozygosity(He)was 0.880,and the average polymorphic information content was 0.846.All sites were highly polymorphic(PIC>0.50).

High-throughput sequencing analysis of differentially expressed mi RNAs and target genes in ischemia/reperfusion injury and apelin-13 neuroprotection

miRNAs regulate a variety of biological processes through pairing-based regulation of gene expression at the 3＇ end of the noncoding region of the target miRNA, miRNAs were found to be abnormally expressed in ischemia/reperfusion injury models. High-throughput sequencing is a recently developed method for sequencing miRNAs and has been widely used in the analysis of miRNAs. In this study, ischemia/reperfusion injury models were intracerebroventricularly injected with 50 pg/kg apelin-13. High-throughput sequencing showed that 357 known miRNAs were differentially expressed among rat models, among which 78 changed to 〉 2-fold or 〈 0.5-fold. Quantita- tive real-time polymerase chain reaction was selected to confirm the expression levels of four miRNAs that were differentially expressed, the results of which were consistent with the results of high-throughput sequencing. Gene Ontology analysis revealed that the predicted targets of the different miRNAs are particularly associated with cellular process, metabolic process, single-organism process, cell, and binding. Kyoto Encyclopedia of Gene and Genome analysis showed that the target genes are involved in metabolic pathways, mitogen-ac- tivated protein kinase signaling pathway, calcium signaling pathway, and nuclear factor-KB signaling pathway. Our findings suggest that differentially expressed miRNAs and their target genes play an important role in ischemia/reperfusion injury and neuroprotection by apelin-13.

Gelatin filter capture-based high-throughput sequencing analysis of microbial diversity in haze particulate matter

Airborne particulate matter(PM),especially PM2.5,can be easily adsorbed by human respiratory system.Their roles in carrying pathogens for spreading epidemic diseases has attracted great concern.Herein,we developed a novel gelatin filter-based and culture-independent method for investigation of the microbial diversity in PM samples during a haze episode in Tianjin,China.This method involves particle capture by gelatin filters,filter dissolution for DNA extraction,and high-throughput sequencing for analysis of the microbial diversity.A total of 584 operational taxonomic units(OTUs)of bacteria and 370 OTUs of fungi at the genus level were identified during hazy days.The results showed that both bacterial and fungal diversities could be evaluated by this method.This study provides a convenient strategy for investigation of microbial biodiversity in haze,facilitating accurate evaluation of airborne epidemic diseases.

Genome-wide identification of RNA editing in seven porcine tissues by matched DNA and RNA high-throughput sequencing

Background: RNA editing is a co/posttranscriptional modification mechanism that increases the diversity of transcripts, with potential functional consequences. The advent of next-generation sequencing technologies has enabled the identification of RNA edits at unprecedented throughput and resolution. However, our knowledge of RNA editing in swine is still limited.Results: Here, we utilized RES-Scanner to identify RNA editing sites in the brain, subcutaneous fat, heart, liver,muscle, lung and ovary in three 180-day-old Large White gilts based on matched strand-specific RNA sequencing and whole-genome resequencing datasets. In total, we identified 74863 editing sites, and 92.1% of these sites caused adenosine-to-guanosine(A-to-G) conversion. Most A-to-G sites were located in noncoding regions and generally had low editing levels. In total, 151 A-to-G sites were detected in coding regions(CDS), including 94 sites that could lead to nonsynonymous amino acid changes. We provide further evidence supporting a previous observation that pig transcriptomes are highly editable at PRE-1 elements. The number of A-to-G editing sites ranged from 4155(muscle) to 25001(brain) across the seven tissues. The expression levels of the ADAR enzymes could explain some but not all of this variation across tissues. The functional analysis of the genes with tissuespecific editing sites in each tissue revealed that RNA editing might play important roles in tissue function.Specifically, more pathways showed significant enrichment in the fat and liver than in other tissues, while no pathway was enriched in the muscle.Conclusions: This study identified a total of 74863 nonredundant RNA editing sites in seven tissues and revealed the potential importance of RNA editing in tissue function. Our findings largely extend the porcine editome and enhance our understanding of RNA editing in swine.

Microbial diversity in Huguangyan Maar Lake of China revealed by high-throughput sequencing

Huguangyan Maar Lake is a typical maar lake in the southeast of China. It is well preserved and not disturbed by anthropogenic activities. In this study, microbial community structures in sediment and water samples from Huguangyan Maar Lake were investigated using a high-throughput sequencing method. We found significant differences between the microbial community compositions of the water and the sediment. The sediment samples contained more diverse Bacteria and Archaea than did the water samples. Actinobacteria, Betaproteobacteria, Cyanobacteria, and Deltaproteobacteria predominated in the water samples while Deltaproteobacteria, Anaerolineae, Nitrospira, and Dehalococcoidia were the major bacterial groups in the sediment. As for Archaea, Woesearchaeota (DHVEG-6), unclassified Archaea, and Deep Sea Euryarchaeotic Group were detected at higher abundances in the water, whereas the Miscellaneous Crenarchaeotic Group, Thermoplasmata, and Methanomicrobia were significantly more abundant in the sediment. Interactions between Bacteria and Archaea were common in both the water column and the sediment. The concentrations of major nutrients (NO^3-, PO4^3-, SiO3^2- and NH4^+) shaped the microbial population structures in the water. At the higher phylogenetic levels including phylum and class, many of the dominant groups were those that were also abundant in other lakes;however, novel microbial populations (unclassified) were often seen at the lower phylogenetic levels. Our study lays a foundation for examining microbial biogeochemical cycling in sequestered lakes or reservoirs.

Characterization of Bacterial Community Associated with Four Organs of the Yesso Scallop (Patinopecten yessoensis) by High-Throughput Sequencing

We used Illumina high-throughput sequencing of PCR-amplified V3-V4 16 S rRNA gene regions to characterize bacterial communities associated with the adductor muscles, gills, gonads and intestines of the Yesso scallop(Patinopecten yessoensis) from waters around Zhangzidao, Dalian, China. Overall, 421,276 optimized reads were classified as 25 described bacterial phyla and 308 genera. Firmicutes, Proteobacteria, Tenericutes, Bacteroidetes, Chlamydiae and Spirochaetae accounted for > 97% of the total reads in the four organs. The bacterial 16 S rDNA sequences assigned to Firmicutes and Proteobacteria were abundant in the adductor muscles, gills and gonads; while reads from Tenericutes were dominant in the intestines, followed by those from Firmicutes, Chlamydiae, Proteobacteria and Bacteroidetes. At the genus level, the dominant genera in the adductor muscles, gills and gonads appeared to be Bacillus, Enterococcus and Lactococcus, whereas Mycoplasma was dominant in the intestines. The relative abundances of Bacillus, Enterococcus, Lactococcus, Alkaliphilus, Raoultella, Paenibacillus and Oceanobacillus were significantly lower in the intestine than in the other three organs. Cluster analysis and principal coordinates analysis of the operational taxonomy units profile revealed significant differences in the bacterial community structure between the intestine and the other three organs. Taken together, these results suggest that scallops have intestine-specific bacterial communities and the adductor muscles, gills and gonads harbor similar communities. The difference in the bacterial community between organs may relate to unique habitats, surroundings, diet and their respective physiological functions.

High-throughput sequencing of highbush blueberry transcriptome and analysis of basic helix-loop-helix transcription factors

The highbush blueberry（Vaccinium corymbosum）,Duke,was used to construct a de novo transcriptome sequence library and to perform data statistical analysis.Mega 4,CLC Sequence Viewer 6 software,and quantitative PCR were employed for bioinformatics and expression analyses of the basic helix-loop-helix（BHLH）transcription factors of the sequencing library.The results showed that 28.38 gigabytes of valid data were obtained from transcriptome sequencing and were assembled into 108 033 unigenes.Functional annotation showed that 32 244 unigenes were annotated into Clusters of Orthologous Groups（COG）and Gene Ontology（GO）databases,whereas the rest of the 75 789 unigenes had no matching information.By using COG and GO classification tools,sequences with annotation information were divided into 25 and 52 categories,respectively,which involved transport and metabolism,transcriptional regulation,and signal transduction.Analysis of the transcriptome library identified a total of 59 BHLH genes.Sequence analysis revealed that 55 genes of that contained a complete BHLH domain.Furthermore,phylogenetic analysis showed that BHLH genes of blueberry（Duke）could be divided into 13 sub-groups.PCR results showed that 45 genes were expressed at various developmental stages of buds,stems,leaves,flowers,and fruits,suggesting that the function of BHLH was associated with the development of different tissues and organs of blueberry,Duke.The present study would provided a foundation for further investigations on the classification and functions of the blueberry BHLH family.

High-throughput sequencing of 16S r RNA amplicons characterizes gut microbiota shift of juvenile sea cucumber Apostichopus japonicus feeding with three antibiotics

Sea cucumber Apostichopus japonicus is an important marine economic species in Asian countries due to its profound nutritional and medicinal value. So far, with the rapid development of intensifi ed artifi cial aquaculture of sea cucumbers, the use of antibiotics is still an inexpensive and dispensable way to treat pathogenic infections, especially during the nursery phase. However, there is little information on the eff ects of antibiotics on the intestinal microbiota of sea cucumber. Therefore an Illumina based sequencing method was used to examine the intestinal bacterial composition of juvenile A . japonicas following diets with three typical antibiotics (tetracycline, erythromycin, and norfl oxacin) under 15, 30, and 45 d. The fi ndings reveal that diff erent antibiotics have distinct eff ects on the growth performance of juvenile sea cucumbers. However, the richness and diversity of microbiota were barely aff ected by antibiotics but the community composition alterations indicated that the three antibiotics exhibited their respective patterns of reshaping the intestinal bacteria of juvenile sea cucumbers. In common, the abundance of some sensitive genera with helpful functions, such as Thalassotalea , Shewanella , Sulfi tobacter , and Halomonas decreased signifi cantly with exposure to antibiotics and the abundance of multiple potential pathogenic- and suspected antibiotic-resistant microorganisms like Arcobacter , Leucothrix , and Clostridium_sensu_stricto_1 was found increased signifi cantly in the antibiotic groups. These results suggest that low doses of antibiotics could aff ect the composition of the intestinal microbiota of sea cucumbers and might increase the risk of infection of the hosts. This study could help us to explore how antibacterial compounds modify the gut microbiota of sea cucumbers and provide theoretical guidance in hatchery management by scientifi c antibiotic use in sea cucumber mariculture.

Relationship between hyperlipidemia and the gut microbiome of rats, characterized using high-throughput sequencing

Objective:To determine the effects of a high-fat diet(HFD)on the gut microbiome in rats,to explore the relationship between the intestinal flora and blood lipid profile.Methods:SpragueeDawley rats were fed an HFD for four weeks to induce hyperlipidemia,then 16S rRNA sequencing was used to compare the intestinal flora between hyperlipidemic and control diet-fed rats.Results:The microbiome of rats fed an HFD for four weeks differed from that of control diet-fed rats.Bacterial species that were less abundant were most affected by HFD feeding,among which were many pathogenic species,which became significantly more abundant.Eighteen genera were present in significantly different numbers in hyperlipidemic and control rats,more than half of which have been linked to infection and inflammation,or energy intake and obesity.The results indicated a type of stress response of the flora to a high-fat environment.In addition,the age of the rats tended to influence the gut microbial composition.Conclusion:These findings suggest that HFD may induce hyperlipidemia by affecting the gut microbial composition.Changes in the abundance of pro-inflammatory and pathogenic bacteria,and those that influence energy intake and obesity,may be important mediators of this.

High-throughput sequencing analysis of differential microRNA expression in the process of blocking the progression of chronic atrophic gastritis to gastric cancer by Xianglian Huazhuo formula(香连化浊方)

OBJECTIVE:To explore the mechanism of Xianglian Huazhuo formula(香连化浊方,XLHZ)blocking the development of chronic atrophic gastritis(CAG)to gastric cancer(GC)through bioinformatics analysis and in vitro.METHODS:Pathological morphology of gastric mucosa of rats were observed.High-throughput sequencing was used to analyze the miRNA expression profile of gastric mucosa.The miRanda,miRDB and miRWalk databases were used to predict the differential target genes.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed for differential target genes.Real-time quantitative reverse transcription polymerase chain reaction(qRTPCR)was used to verify the differentially expressed miRNAs and target genes.Western blot,EdU,wound healing and flow cytometry were used to observe the effect of XLHZ on epithelial-mesenchymal transition(EMT)markers,proliferation,migration,apoptosis and cell cycle of CAG cells in vitro.RESULTS:A total of five differentially expressed miRNAs and four differential target genes were screened in this study.GO analysis showed that the target genes were enriched in regulation of neuron development,regulation of transcription factor activity and regulation of RNA polymerase.KEGG pathways database differences in gene enrichment of target genes in the Wnt signaling pathway,Phospholipase D signaling pathway and mitogen-activated protein kinase signaling pathway.qRTPCR confirmed that miRNAs and its target genes were consistent with the screening results.In vitro,our study revealed that XLHZ could increase the expression of Ecadherin,decrease the expression of transforming growth factorβ1,vimentin andβ-catenin,inhibite the proliferation and migration of CAG cells,cause cell cycle arrest at G0/G1 and G2/M phase,induce the apoptosis of CAG cells,and prevent the progression of CAG to GC.CONCLUSION:This study provided a new idea for the mechanism of blocking the progression of CAG to GC by XLHZ,which may be related to the expression of miR-20a-3p,miR-320-3p,miR-34b-5p,miR-483-3p and miR-883-3p and their target genes transferrin receptor,nuclear receptor subfamily 4 member 2,delta like canonical Notch ligand 1 and a kinase anchor protein 12 in CAG.In the future,we will continue to investigate the linkage between the active ingredients of XLHZ and the relevant miRNAs and their target genes,so as to provide more sufficient experimental basis for clinically effective prevention of CAG to GC.

Peripheral blood transcriptome analysis of patients with ovarian hyperstimulation syndrome through high-throughput sequencing

Objective:Ovarian hyperstimulation syndrome(OHSS)is a frequent iatrogenic complication that arises during assisted reproduction and accounts for approximately 30%of all in vitro fertilization cycles.Using high-throughput sequencing,we investigated the peripheral blood transcriptome of patients with OHSS.Methods:Peripheral blood samples were obtained from 15 patients in each of the OHSS high-risk and low-risk groups on the ovum pick-up day.Subsequently,high-throughput sequencing was used to obtain the peripheral blood transcriptomes of five patients each from the high-and low-risk groups.Bioinformatic tools were used for mRNA expression profile mapping and screening of differentially expressed genes(DEGs).Bioinformatics techniques were also implemented in the Kyoto Encyclopedia of Genes Genomes(KEGG)signal pathway,Gene Ontology(GO)function,and protein-protein interaction(PPI)network analyses of DEGs.Results:A total of 20,031 genes were identified and 148 were found to be differentially expressed(P<0.05,|log_(2)FC|>0.58),with 52 upregulated and 96 downregulated genes.GO and KEGG analyses indicated that these genes were involved in extracellular corpuscles(GO:0070062),plasma membrane(GO:0005886),extracellular regions(GO:0005576),immune system response(GO:0006955),PI3K-Akt signaling pathways(hsa04151),cell adhesion molecules(CAMs,hsa04514),focal adhesion(hsa04510),and complement and coagulation cascades(hsa04610).The PPI network and realtime fluorescence quantitative polymerase chain reaction(qPCR)verification predicted that complement C3,von Willebrand factor,and vascular cell adhesion protein 1 proteins are highly implicated in OHSS and may serve as potential biomarkers for future OHSS studies.Conclusion:Transcriptome analysis revealed several DEGs related to OHSS risk factors in the peripheral blood,indicating that these DEGs may be novel players in OHSS development.

Single‑cell RNA sequencing opens a new era for cotton genomic research and gene functional analysis

Single-cell RNA sequencing(scRNA-seq)is one of the most advanced sequencing technologies for studying transcriptome landscape at the single-cell revolution.It provides numerous advantages over traditional RNA-seq.Since it was first used to profile single-cell transcriptome in plants in 2019,it has been extensively employed to perform different research in plants.Recently,scRNA-seq was also quickly adopted by the cotton research community to solve lots of scientific questions which have been never solved.In this comment,we highlighted the significant progress in employing scRNA-seq to cotton genetic and genomic study and its future potential applications.