The 16S rRNA gene is the most commonly used molecular marker for identifying microorganisms. It is used in sequencing technology, including the first-generation, the second-generation, and the third-generation sequencing technology. A large number of studies on the 16S rRNA gene have contributed to a deeper understanding of oral microbial diversity. In the healthy oral cavity, there is microbial diversity in time and space. With the occurrence or development of oral diseases such as caries, periodontal disease, or halitosis, the microbial diversity will be changed.
High-Throughput Nucleotide Sequencing ; Mouth ; RNA, Ribosomal, 16S ; genetics

RNA is a polymeric molecule implicated in various biological processes, such as the coding, decoding, regulation, and expression of genes. Numerous studies have examined RNA features using whole transcriptome sequencing (RNA-seq) approaches. RNA-seq is a powerful technique for characterizing and quantifying the transcriptome and accelerates the development of bioinformatics software. In this review, we introduce routine RNA-seq workflow together with related software, focusing particularly on transcriptome reconstruction and expression quantification.
Biological Processes ; Clinical Coding ; Computational Biology ; Gene Expression ; High-Throughput Nucleotide Sequencing ; Polymers ; RNA ; Transcriptome*

BACKGROUNDData from RNA-seq experiments provide a wealth of information about the transcriptome of an organism. However, the analysis of such data is very demanding. In this study, we aimed to establish robust analysis procedures that can be used in clinical practice.

METHODSWe studied RNA-seq data from triple-negative breast cancer patients. Specifically, we investigated the subsampling of RNA-seq data.

RESULTSThe main results of our investigations are as follows: (1) the subsampling of RNA-seq data gave biologically realistic simulations of sequencing experiments with smaller sequencing depth but not direct scaling of count matrices; (2) the saturation of results required an average sequencing depth larger than 32 million reads and an individual sequencing depth larger than 46 million reads; and (3) for an abrogated feature selection, higher moments of the distribution of all expressed genes had a higher sensitivity for signal detection than the corresponding mean values.

CONCLUSIONSOur results reveal important characteristics of RNA-seq data that must be understood before one can apply such an approach to translational medicine.

Microbes are the most important commensal organisms in humans, animals and plants, and are the major habitants in soil, sediment, water, air and other habitats. The analysis of microbiome in these habitats has become a basic research technique. As a fast developing technology in recent years, microbiome sequencing and analysis have been widely used in human health, environmental pollution control, food industry, agriculture and animal husbandry and other fields. In order to sort out and summarize the current status, development and application prospects of microbiome sequencing and analysis technologies, this special issue has prepared a collection of 16 papers in this field, that comprise sample preservation and processing, single microbe genome sequencing and analysis, and microbiome feature analysis in special habitats, microbiome related databases and algorithms, and microbiome sequencing and analysis expert consensus. It also introduced in detail the development trend of the microbiome sequencing and analysis, in order to promote the rapid development of the microbiome sequencing and analysis industry and scientific research in China, and provide necessary reference for the healthy development of related industries.
Animals ; Bacteria/genetics* ; China ; High-Throughput Nucleotide Sequencing ; Humans ; Metagenome ; Microbiota/genetics* ; RNA, Ribosomal, 16S

This study aims to identify the circular RNAs (circRNAs) in the liver of whitespotted bamboo shark (Chiloscyllium plagiosum) and to explore the effect of the overexpression of circRNAs on the proliferation and migration of hepatocellular carcinoma HepG2 cells. We conducted high-throughput sequencing for prediction of the circRNAs and then designed forward and reverse primers to verify them. Further, we constructed overexpression vectors for transient transfection of circRNAs into HepG2 cells. Finally, we employed CCK-8 assay and scratch assay to measure the proliferation and migration of the treated HepG2 cells. A total of 4 558 circRNAs were predicted, among which 14 circRNAs were confirmed. The qRT-PCR showed that circRNA 13-566, circRNA 4-475, circRNA 5-402, circRNA 294-177, and circRNA 30-219 were transiently overexpressed in HepG2 cells. The overexpression of these five circRNAs inhibited the proliferation and migration of HepG2 cells to varying degrees, and circRNA 4-475 and circRNA 294-177 had especially notable effect. This study provided a basic database of circRNA genes that particularly active in whitespotted bamboo shark liver and demonstrated with functional studies of these circRNAs potentially involved in normal and malignant liver cells.
Animals ; High-Throughput Nucleotide Sequencing ; Liver ; RNA, Circular/genetics* ; Sharks/genetics* ; Sincalide/genetics*

Objective To preliminarily discuss the feasibility of geolocation inference of forensic individual origin by soil metagenomic analysis. Methods The 33 soil samples from Heilongjiang, Qinghai and Tibet were collected, total bacterial DNA in the samples were extracted, and universal primers were used to amplify the V3 and V4 hypervariable region of bacterial 16S rDNA. The region was sequenced by high-throughput sequencing （HTS） with the MiSeq sequencer. Bioinformatics analysis such as species composition and sample comparison was performed on sequencing data. The richness index and diversity index were calculated based on operational taxonomic unit （OTU） results. Results A total of 2 720 149 sequences were generated by sequencing. Those sequences were clustered into 114 848 OTUs. The Chao1 indexes of soil microorganisms in Heilongjiang, Qinghai, and Tibet were 797.45, 745.11 and 535.98, respectively, and Shannon indexes were 6.46, 6.36 and 6.25, respectively. The number of bacterial species and the community diversity in the soil from high to low were Heilongjiang > Qinghai > Tibet. The composition of soil bacteria in three provinces at various classification levels were obtained, the dominant genuses in Heilongjiang were Chthoniobacteraceae DA101 and an unannotated genus of Thermogemmatisporaceae; the dominant genuses in Qinghai were an unannotated genus of Cytophagaceae and an unannotated genus of Nocardioidaceae; the dominant genuses in Tibet were an unannotated genus of Comamonadaceae and Verrucomicrobiaceae Luteolibacter. The results of principal co-ordinates analysis demonstrated that, according to the weighted UniFrac analysis, the three principle components represented 56.36% of the total variable, and according to the unweighted UniFrac analysis, the three principle components represented 34.81% of the total variable. The samples from the same province could be clustered together, and the species and content of soil microorganisms from different provinces were significantly different. Conclusion Based on the metagenomic analysis method, soil samples from different regions can be effectively distinguished, which has potential application value in geolocation inference of forensic individual origin in the future.
Bacteria/genetics* ; High-Throughput Nucleotide Sequencing ; RNA, Ribosomal, 16S/genetics* ; Soil ; Soil Microbiology

OBJECTIVE: Exosomal long noncoding RNAs (lncRNAs) are the key to diagnosing and treating various diseases. This study aimed to investigate the diagnostic value of plasma exosomal lncRNAs in white matter hyperintensities (WMH). METHODS: We used high-throughput sequencing to determine the differential expression (DE) profiles of lncRNAs in plasma exosomes from WMH patients and controls. The sequencing results were verified in a validation cohort using qRT-PCR. The diagnostic potential of candidate exosomal lncRNAs was proven by binary logistic analysis and receiver operating characteristic (ROC) curves. The diagnostic value of DE exo-lncRNAs was determined by the area under the curve (AUC). The WMH group was then divided into subgroups according to the Fazekas scale and white matter lesion site, and the correlation of DE exo-lncRNAs in the subgroup was evaluated. RESULTS: In our results, four DE exo-lncRNAs were identified, and ROC curve analysis revealed that exo-lnc_011797 and exo-lnc_004326 exhibited diagnostic efficacy for WMH. Furthermore, WMH subgroup analysis showed exo-lnc_011797 expression was significantly increased in Fazekas 3 patients and was significantly elevated in patients with paraventricular matter hyperintensities. CONCLUSION Plasma exosomal lncRNAs have potential diagnostic value in WMH. Moreover, exo-lnc_011797 is considered to be a predictor of the severity and location of WMH.
Humans ; RNA, Long Noncoding/genetics* ; White Matter ; Area Under Curve ; Exosomes/genetics* ; High-Throughput Nucleotide Sequencing

RNA-binding protein exerts important biological function by specifically recognizing RNA motif. SELEX (Systematic evolution of ligands by exponential enrichment), an in vitro selection method, can obtain consensus motif with high-affinity and specificity for many target molecules from DNA or RNA libraries. Here, we combined SELEX with next-generation sequencing to study the protein-RNA interaction in vitro. A pool of RNAs with 20 bp random sequences were transcribed by T7 promoter, and target protein was inserted into plasmid containing SBP-tag, which can be captured by streptavidin beads. Through only one cycle, the specific RNA motif can be obtained, which dramatically improved the selection efficiency. Using this method, we found that human hnRNP A1 RRMs domain (UP1 domain) bound RNA motifs containing AGG and AG sequences. The EMSA experiment indicated that hnRNP A1 RRMs could bind the obtained RNA motif. Taken together, this method provides a rapid and effective method to study the RNA binding specificity of proteins.
Aptamers, Nucleotide ; Gene Library ; Heterogeneous Nuclear Ribonucleoprotein A1 ; genetics ; High-Throughput Nucleotide Sequencing ; Humans ; RNA ; SELEX Aptamer Technique

BACKGROUND: Trichorhinophalangeal syndrome (TRPS) patients tend to have alopecia that appears to be androgenetic, and this genetic model might give clues to the pathogenesis of hair loss or hair morphogenesis. OBJECTIVE: This study was conducted to identify additional genetic evidence of TRPS and hair morphogenesis from a TRPS patient. METHODS: From one TRPS type I patient, we extracted RNA and profiled whole transcriptome in non-balding and balding scalp areas using high-throughput RNA sequencing. RESULTS: We found a total of 26,320 genes, which comprised 14,892 known genes with new isoforms and 4,883 novel genes from the non-balding and balding areas. Among these, a total of 1,242 genes showed different expression in the two scalp areas (p<0.05 and log2 fold-change >0). Several genes related to the skin and hair, alopecia, and the TRPS1 gene were validated by qRT-PCR. Twelve of 15 genes (KRT6C, KRTAP3-1, MKI67, GPRC5D, TYRP1, DSC1, PMEL, WIF1, SOX21, TINAG, PTGDS, and TRPS1) were down-regulated (10 genes: p<0.01; SOX21 and PTGDS: p>0.05), and the three other genes (HBA2, GAL, and DES) were up-regulated (p<0.01) in the balding scalp. Many genes related to keratin and hair development were down-regulated in the balding scalp of the TRPS type I patient. In particular, the TRPS1 gene might be related to androgen metabolism and hair morphogenesis. CONCLUSION: Our result could suggest a novel perspective and evidence to support further study of TRPS and hair morphogenesis.
Alopecia ; Gene Expression Profiling* ; Hair ; High-Throughput Nucleotide Sequencing ; Humans ; Metabolism ; Models, Genetic ; Morphogenesis ; Protein Isoforms ; RNA ; Scalp* ; Skin ; Transcriptome*

In recent years, 16S rRNA amplicon sequencing technology has been widely used to study human gut microbiota and to detect unknown pathogens in clinical samples. However, its resolution to bacterial population can only reach the relative abundance of genus level, and different factors affect the final bacterial profile, such as sample concentrations, PCR cycle numbers and amplification primers. In order to solve these problems, we developed a quantitative 16S rRNA amplicon sequencing method by combining random tag and internal marker method. The new methods improved the accuracy of human gut microbiota, reduced the impact of experimental operation on the results, and improved the comparability between sequencing and other molecular biological methods.
Bacteria/genetics* ; Gastrointestinal Microbiome/genetics* ; High-Throughput Nucleotide Sequencing ; Humans ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S/genetics*