OBJECTIVES: Uterine fluid RNA can be used as a test for endometrial receptivity, but there is still no noninvasive sampling method available. The polyvinyl alcohol (PVA) formaldehyde absorbent sponge, a medical bio-absorbent sponge with good water absorption and biophilic properties, can be used to develop a new noninvasive endometrial fluid sampler. This study aims to investigate the toxicity of PVA acetal absorbent sponges on endometrial epithelial cells and its effect on RNA sequencing (RNA-Seq). METHODS: The experimental group using PVA formaldehyde absorbent sponge was prepared into 0.005%, 0.01% and 0.02% (w/v) suspension, and 0.01%, 0.05% and 0.1% (v/v) extract groups. The control group was only the complete culture medium. Nothing was added to the blank group. In vitro cytotoxicity assay was used to evaluate the survival rate of cells. Eight patients underwent in vitro fertilization treatment in the Reproductive Center of Xiangya Hospital, Central South University from November 2019 to January 2020. The uterine fluid of each patient was aspirated. The experimental group was inhaled with sterile PVA formaldehyde absorbent sponge and then immersed RNA-later solution. The control group was directly injected into the same amount of RNA-later solution. RNA-seq and data analysis was performed later. RESULTS: The vitro cytotoxicity assay showed that in suspension groups, there was no significance difference in cell survival between different co-culture time in 0.005% group (P=0.255). In the 0.01% and 0.02% group, there was no difference at each incubation time within 12 h (all P>0.05), but the cell survival rate was decreased at 24 h compared with 0 h (P<0.01, P<0.05). At the same co-culture time, the cell survival of the 3 concentration gradient groups were significantly lower than that of the control group (all P<0.05). The cell viability of the 0.005% concentration group was decreased less than 30% at 24 h, the 0.01% concentration group decreased more than 30% at 12 h, and the 0.02% concentration group was decreased more than 30% at 0 h. For extract groups, there was no significant difference in the survival rate within 6 h in 0.01% concentration group (all P>0.05), and the survival rate of 12 h and 24 h was lower than that of 0 h group (both P<0.01). In 0.05% group, there was no significant difference at each incubation time within 12 h (all P>0.05), but the survival rate at 24 h was lower than that at 0 h (P<0.05). There was no significant difference in survival rate at different culture time in 0.1% concentration group (P=0.082). At the same culture time, there was no significant difference in survival rate between 0.01% group and control group at 0, 3 and 24 h (all P>0.05). Except for 3 h, the survival rate of 0.05% and 0.1% groups was lower than that of control group (all P<0.05), and the decrease was all less than 30%. Uterine fluid RNA-seq showed that there was no significance difference in exonic rate, the detected genes and transcripts of RNA between the experiment groups and the control group (all P>0.05). CONCLUSIONS The in vitro cytotoxic of PVA formaldehyde absorbent sponge on human endometrial epithelial cell meet the national standard of the cytotoxic of medical materials. Sampling the uterine fluid with this material does not affect the RNA-Seq results. PVA formaldehyde absorbent sponge is safe and feasible when appling to the noninvasive uterine fluid sampling and RNA sequencing.
Humans ; Social Group ; Sequence Analysis, RNA ; RNA

With the rapid development of molecular biotechnology, transcriptomics has been widely used in the study of gene expression. In recent years, the techniques for microbial transcriptomics research have also been rapidly developing. At the gene level, the way for obtaining sequence information has been developed from complementary validation of RNA fragment through DNA microarray to direct sequencing of full-length RNA. Spatially, the traditional population transcriptomics technique has been developed into spatial, single cell and epigenetic transcriptomics studies. With the application of transcriptomics techniques in the field of microbial research, the corresponding defects were gradually revealed and constantly improved. In this paper, the traditional and new transcriptomics techniques in the field of microbial research are summarized to provide reference for microbial transcriptomics research.
Transcriptome ; Oligonucleotide Array Sequence Analysis ; Sequence Analysis ; RNA ; Biotechnology

The MinION(TM) is a miniature nanopore-based analysis device in which the characteristics of an analyte, as it passes through the nanopore, cause changes in the flow of ions through the pore, which are measured, as current flow, by a low noise amplifier and analogue-to-digital converter. Potentially any molecular analyte capable of passing through the nanopore may modify the flow of ions and generate a signal which might be diagnostic. In practice the current device is focussed on DNA sequencing, directly sequencing RNA is a likely development. With the MinION Access Program making the MinION(TM) widely available a flood of applications exploiting its real time, long read capabilities have been published. We review the background to the technology and compare it to current next generation sequencing.
Ions ; Nanopores ; Noise ; RNA ; Sequence Analysis, DNA

To identify pathways associated with survival phenotypes using gene expression data, we recently proposed the hierarchical structural component model for pathway analysis of gene expression data (HisCoM-PAGE) method. The HisCoM-PAGE software can consider hierarchical structural relationships between genes and pathways and analyze multiple pathways simultaneously. It can be applied to various types of gene expression data, such as microarray data or RNA sequencing data. We expect that the HisCoM-PAGE software will make our method more easily accessible to researchers who want to perform pathway analysis for survival times.
Gene Expression ; Methods ; Phenotype ; Sequence Analysis, RNA

Currently, Illumina sequencers are the globally leading sequencing platform in the next-generation sequencing market. Recently, MGI Tech launched a series of new sequencers, including the MGISEQ-2000, which promise to deliver high-quality sequencing data faster and at lower prices than Illumina’s sequencers. In this study, we compared the performance of two major sequencers (MGISEQ-2000 and HiSeq 4000) to test whether the MGISEQ-2000 sequencer delivers high-quality sequence data as suggested. We performed RNA sequencing of four human colon cancer samples with the two platforms, and compared the sequencing quality and expression values. The data produced from the MGISEQ-2000 and HiSeq 4000 showed high concordance, with Pearson correlation coefficients ranging from 0.98 to 0.99. Various quality control (QC) analyses showed that the MGISEQ-2000 data fulfilled the required QC measures. Our study suggests that the performance of the MGISEQ-2000 is comparable to that of the HiSeq 4000 and that the MGISEQ-2000 can be a useful platform for sequencing.
Benchmarking ; Colonic Neoplasms ; Humans ; Quality Control ; RNA ; Sequence Analysis, RNA

Forensic microorganism is one of the hotspots of forensic science research. Due to its conservatism and specificity, the 16S rRNA gene is found to be an ideal marker for forensic identification. With the rapid development of high throughput sequencing technology, the research on microorganisms has been gradually applied to many fields such as environment and health care. In the field of forensic science, the results of forensic microbiology research, represented by 16S rRNA gene sequencing, are also gradually applied to forensic practice, such as biological samples identification, individual identification, postmortem interval estimation, and regional inference, which not only provide clues for the investigation of cases but also complement and assist traditional methods. This paper describes the research methods and related sequencing technologies of 16S rRNA gene sequencing, summarizes its research progress, and discusses the application value and potential of 16S rRNA in forensic science.
Forensic Sciences/trends* ; RNA, Ribosomal, 16S ; Sequence Analysis, RNA

Traditional bulk RNA sequencing assesses the average expression level of genes in tissues rather than the differences in cellular responses. Accordingly, it is hard to differentiate sensitive responding cells, leading to inaccurate identification of toxicity pathways. Single-cell RNA sequencing (scRNA-seq) isolated single cells from tissue and subjected them to cell subtypes-specific transcriptome analysis. This technique in toxicological studies realizes the heterogeneous cellular responses in the tissue microenvironment upon chemical exposure. Thus it helps to identify sensitive responding cells and key molecular events, providing a powerful tool and a new perspective for exploring the mechanisms of toxicity and the modes of action. This review summarizes the development, principle, method, application and limitations of scRNA-seq in mechanistic toxicological researches, and discusses the prospect of multi-directional applications.
Base Sequence ; Gene Expression Profiling ; Sequence Analysis, RNA ; Single-Cell Analysis ; Transcriptome

The emergence of single-cell sequencing technology enables people to observe cells with unprecedented precision. However, it is difficult to capture the information on all cells and genes in one single-cell RNA sequencing (scRNA-seq) experiment. Single-cell data of a single modality cannot explain cell state and system changes in detail. The integrative analysis of single-cell data aims to address these two types of problems. Integrating multiple scRNA-seq data can collect complete cell types and provide a powerful boost for the construction of cell atlases. Integrating single-cell multimodal data can be used to study the causal relationship and gene regulation mechanism across modalities. The development and application of data integration methods helps fully explore the richness and relevance of single-cell data and discover meaningful biological changes. Based on this, this article reviews the basic principles, methods and applications of multiple scRNA-seq data integration and single-cell multimodal data integration. Moreover, the advantages and disadvantages of existing methods are discussed. Finally, the future development is prospected.
Base Sequence ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Sequence Analysis, RNA ; Single-Cell Analysis

Prediction of RNA secondary structures including pseudoknots is a difficult topic in RNA field. Current predicting methods usually have relatively low accuracy and high complexity. Considering that the stacking of adjacent base pairs is a common feature of RNA secondary structure, here we present a method for predicting pseudoknots based on covariance with stacking and minimum free energy. A new score scheme, which combined stacked covariance with free energy, was used to assess the evaluation of base pair in our method. Based on this score scheme, we utilized an iterative procedure to compute the optimized RNA secondary structure with minimum score approximately. In each interaction, helix of high covariance and low free energy was selected until the sequences didn't form helix, so two crossing helixes which were selected from different iterations could form a pseudoknot. We test our method on data sets of ClustalW alignments and structural alignments downloaded from RNA databases. Experimental results show that our method can correctly predict the major portion of pseudoknots. Our method has both higher average sensitivity and specificity than the reference algorithms, and performs much better for structural alignments than for ClustalW alignments. Finally, we discuss the influence on the performance by the factor of covariance weight, and conclude that the best performance is achieved when lambda1 : lambda2 = 5 : 1.
Algorithms ; Base Pairing ; Base Sequence ; Computational Biology ; methods ; Molecular Sequence Data ; Nucleic Acid Conformation ; RNA ; chemistry ; genetics ; Sequence Analysis, RNA

The rhizome of Panax japonicus var. major have been used as the natural medicinal agent by Chinese traditional doctors for more than thousand years. Most of the therapeutic effects of P. japonicus var. major had been reported due to the presence of tetracyclic or pentacyclic triterpene saponins. In this study, Illumina pair-end RNA-sequencing and de novo splicing were done in order to understand the pathway of triterpenoid saponins in this species. The valid reads data of 15. 6 Gb were obtained. The 62 240 unigenes were finally obtained by de novo splicing. After annotation, we discovered 19 unigenes involved in ginsenoside backbone biosynthesis. Additionally, 69 unigenes and 18 unigenes were predicted to have potential function of cytochrome P450 and UDP-glycosyltransferase based on the annotation results, which may encode enzymes responsible for ginsenoside backbone modification. This study provides global expressed datas for P. japonicus var. major, which will contribute significantly to further genome-wide research and analysis for this species.
Gene Expression Profiling ; Panax ; genetics ; Saponins ; biosynthesis ; Sequence Analysis, RNA