-
International Journal of Molecular... Jun 2024RNA sequencing (RNA-Seq) is a powerful technique and is increasingly being used in clinical research and drug development. Currently, several RNA-Seq methods have been... (Comparative Study)
Comparative Study
RNA sequencing (RNA-Seq) is a powerful technique and is increasingly being used in clinical research and drug development. Currently, several RNA-Seq methods have been developed. However, the relative advantage of each method for degraded RNA and low-input RNA, such as RNA samples collected in the field of clinical setting, has remained unknown. The Standard method of RNA-Seq captures mRNA by poly(A) capturing using Oligo dT beads, which is not suitable for degraded RNA. Here, we used three commercially available RNA-Seq library preparation kits (SMART-Seq, xGen Broad-range, and RamDA-Seq) using random primer instead of Oligo dT beads. To evaluate the performance of these methods, we compared the correlation, the number of detected expressing genes, and the expression levels with the Standard RNA-Seq method. Although the performance of RamDA-Seq was similar to that of Standard RNA-Seq, the performance for low-input RNA and degraded RNA has decreased. The performance of SMART-Seq was better than xGen and RamDA-Seq in low-input RNA and degraded RNA. Furthermore, the depletion of ribosomal RNA (rRNA) improved the performance of SMART-Seq and xGen due to increased expression levels. SMART-Seq with rRNA depletion has relative advantages for RNA-Seq using low-input and degraded RNA.
Topics: Sequence Analysis, RNA; Humans; RNA Stability; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; RNA; RNA, Ribosomal; RNA, Messenger; RNA-Seq
PubMed: 38892331
DOI: 10.3390/ijms25116143 -
International Journal of Molecular... Jun 2024The microbiome of the ocular surface has been characterised, but only limited information is available on a possible silent intraocular microbial colonisation in normal...
The microbiome of the ocular surface has been characterised, but only limited information is available on a possible silent intraocular microbial colonisation in normal eyes. Therefore, we performed next-generation sequencing (NGS) of 16S rDNA genes in the aqueous humour. The aqueous humour was sampled from three patients during cataract surgery. Air swabs, conjunctival swabs from patients as well as from healthy donors served as controls. Following DNA extraction, the V3 and V4 hypervariable regions of the 16S rDNA gene were amplified and sequenced followed by denoising. The resulting Amplicon Sequence Variants were matched to a subset of the Ribosomal Database Project 16S database. The deduced bacterial community was then statistically analysed. The DNA content in all samples was low (0-1.49 ng/µL) but sufficient for analysis. The main phyla in the samples were (48%), (26%), (14%), (8%), and (2%). Patients' conjunctival control samples and anterior chamber fluid showed similar patterns of bacterial species containing many waterborne species. Non-disinfected samples showed a different bacterial spectrum than the air swab samples. The data confirm the existence of an ocular surface microbiome. Meanwhile, a distinct intraocular microbiome was not discernible from the background, suggesting the absence of an intraocular microbiome in normal eyes.
Topics: Humans; Aqueous Humor; Microbiota; High-Throughput Nucleotide Sequencing; RNA, Ribosomal, 16S; Bacteria; DNA, Bacterial; Male; Female; Aged; Middle Aged; Sequence Analysis, DNA
PubMed: 38892316
DOI: 10.3390/ijms25116128 -
International Journal of Molecular... May 2024Muscular atrophy is a complex catabolic condition that develops due to several inflammatory-related disorders, resulting in muscle loss. Tumor necrosis factor alpha...
Muscular atrophy is a complex catabolic condition that develops due to several inflammatory-related disorders, resulting in muscle loss. Tumor necrosis factor alpha (TNF-α) is believed to be one of the leading factors that drive inflammatory response and its progression. Until now, the link between inflammation and muscle wasting has been thoroughly investigated, and the non-coding RNA machinery is a potential connection between the candidates. This study aimed to identify specific miRNAs for muscular atrophy induced by TNF-α in the C2C12 murine myotube model. The difference in expression of fourteen known miRNAs and two newly identified miRNAs was recorded by next-generation sequencing between normal muscle cells and treated myotubes. After validation, we confirmed the difference in the expression of one novel murine miRNA (nov-mmu-miRNA-1) under different TNF-α-inducing conditions. Functional bioinformatic analyses of nov-mmu-miRNA-1 revealed the potential association with inflammation and muscle atrophy. Our results suggest that nov-mmu-miRNA-1 may trigger inflammation and muscle wasting by the downregulation of LIN28A/B, an anti-inflammatory factor in the let-7 family. Therefore, TNF-α is involved in muscle atrophy through the modulation of the miRNA cellular machinery. Here, we describe for the first time and propose a mechanism for the newly discovered miRNA, nov-mmu-miRNA-1, which may regulate inflammation and promote muscle atrophy.
Topics: Animals; MicroRNAs; Mice; Tumor Necrosis Factor-alpha; Muscular Atrophy; Cell Line; Muscle, Skeletal; Muscle Fibers, Skeletal; Gene Expression Regulation; High-Throughput Nucleotide Sequencing
PubMed: 38892252
DOI: 10.3390/ijms25116064 -
International Journal of Molecular... May 2024Jeryak is the F1 generation of the cross between Gannan yak and Jersey cattle, which has the advantages of fast growth and high adaptability. The growth and development...
Jeryak is the F1 generation of the cross between Gannan yak and Jersey cattle, which has the advantages of fast growth and high adaptability. The growth and development of skeletal muscle is closely linked to meat production and the quality of meat. However, the molecular regulatory mechanisms of muscle growth differences between Gannan yak and Jeryak analyzed from the perspective of chromatin opening have not been reported. In this study, ATAC-seq was used to analyze the difference of chromatin openness in longissimus muscle of Gannan yak and Jeryak. It was found that chromatin accessibility was more enriched in Jeryak compared to Gannan yak, especially in the range of the transcription start site (TSS) ± 2 kb. GO and KEGG enrichment analysis indicate that differential peak-associated genes are involved in the negative regulation of muscle adaptation and the Hippo signaling pathway. Integration analysis of ATAC-seq and RNA-seq revealed overlapping genes were significantly enriched during skeletal muscle cell differentiation and muscle organ morphogenesis. At the same time, we screened , , and for possible involvement in skeletal muscle development, constructed a genes and transcription factors network map, and found that some transcription factors (TFs), including YY1, KLF4, KLF5 and Bach1, were involved in skeletal muscle development. Overall, we have gained a comprehensive understanding of the key factors that impact skeletal muscle development in various breeds of cattle, providing new insights for future analysis of the molecular regulatory mechanisms involved in muscle growth and development.
Topics: Animals; Cattle; Muscle, Skeletal; RNA-Seq; Chromatin Immunoprecipitation Sequencing; Muscle Development; Chromatin; Meat; Transcription Factors
PubMed: 38892214
DOI: 10.3390/ijms25116029 -
International Journal of Molecular... May 2024Single-cell RNA sequencing (scRNA-seq) is widely used to interpret cellular states, detect cell subpopulations, and study disease mechanisms. In scRNA-seq data analysis,...
Single-cell RNA sequencing (scRNA-seq) is widely used to interpret cellular states, detect cell subpopulations, and study disease mechanisms. In scRNA-seq data analysis, cell clustering is a key step that can identify cell types. However, scRNA-seq data are characterized by high dimensionality and significant sparsity, presenting considerable challenges for clustering. In the high-dimensional gene expression space, cells may form complex topological structures. Many conventional scRNA-seq data analysis methods focus on identifying cell subgroups rather than exploring these potential high-dimensional structures in detail. Although some methods have begun to consider the topological structures within the data, many still overlook the continuity and complex topology present in single-cell data. We propose a deep learning framework that begins by employing a zero-inflated negative binomial (ZINB) model to denoise the highly sparse and over-dispersed scRNA-seq data. Next, scZAG uses an adaptive graph contrastive representation learning approach that combines approximate personalized propagation of neural predictions graph convolution (APPNPGCN) with graph contrastive learning methods. By using APPNPGCN as the encoder for graph contrastive learning, we ensure that each cell's representation reflects not only its own features but also its position in the graph and its relationships with other cells. Graph contrastive learning exploits the relationships between nodes to capture the similarity among cells, better representing the data's underlying continuity and complex topology. Finally, the learned low-dimensional latent representations are clustered using Kullback-Leibler divergence. We validated the superior clustering performance of scZAG on 10 common scRNA-seq datasets in comparison to existing state-of-the-art clustering methods.
Topics: Single-Cell Analysis; Cluster Analysis; Humans; RNA-Seq; Sequence Analysis, RNA; Algorithms; Software; Deep Learning; Computational Biology; Single-Cell Gene Expression Analysis
PubMed: 38892162
DOI: 10.3390/ijms25115976 -
International Journal of Molecular... May 2024A common result of infection is an abnormal immune response, which may be detrimental to the host. To control the infection, the immune system might undergo regulation,...
A common result of infection is an abnormal immune response, which may be detrimental to the host. To control the infection, the immune system might undergo regulation, therefore producing an excess of either pro-inflammatory or anti-inflammatory pathways that can lead to widespread inflammation, tissue damage, and organ failure. A dysregulated immune response can manifest as changes in differentiated immune cell populations and concentrations of circulating biomarkers. To propose an early diagnostic system that enables differentiation and identifies the severity of immune-dysregulated syndromes, we built an artificial intelligence tool that uses input data from single-cell RNA sequencing. In our results, single-cell transcriptomics successfully distinguished between mild and severe sepsis and COVID-19 infections. Moreover, by interpreting the decision patterns of our classification system, we identified that different immune cells upregulating or downregulating the expression of the genes , , , , , and can accurately differentiate between different degrees of infection. Our research has identified genes of significance that effectively distinguish between infections, offering promising prospects as diagnostic markers and providing potential targets for therapeutic intervention.
Topics: Humans; COVID-19; Machine Learning; RNA-Seq; Biomarkers; SARS-CoV-2; Single-Cell Analysis; Sepsis; Transcriptome; Gene Expression Profiling; Sequence Analysis, RNA; Single-Cell Gene Expression Analysis
PubMed: 38892107
DOI: 10.3390/ijms25115920 -
International Journal of Molecular... May 2024The Epstein-Barr virus (EBV) is frequently found in endomyocardial biopsies (EMBs) from patients with heart failure, but the detection of EBV-specific DNA has not been...
The Epstein-Barr virus (EBV) is frequently found in endomyocardial biopsies (EMBs) from patients with heart failure, but the detection of EBV-specific DNA has not been associated with progressive hemodynamic deterioration. In this paper, we investigate the use of targeted next-generation sequencing (NGS) to detect EBV transcripts and their correlation with myocardial inflammation in EBV-positive patients with heart failure with reduced ejection fraction (HFrEF). Forty-four HFrEF patients with positive EBV DNA detection and varying degrees of myocardial inflammation were selected. EBV-specific transcripts from EMBs were enriched using a custom hybridization capture-based workflow and, subsequently, sequenced by NGS. The short-read sequencing revealed the presence of EBV-specific transcripts in 17 patients, of which 11 had only latent EBV genes and 6 presented with lytic transcription. The immunohistochemical staining for CD3 T lymphocytes showed a significant increase in the degree of myocardial inflammation in the presence of EBV lytic transcripts, suggesting a possible influence on the clinical course. These results imply the important role of EBV lytic transcripts in the pathogenesis of inflammatory heart disease and emphasize the applicability of targeted NGS in EMB diagnostics as a basis for specific treatment.
Topics: Humans; Herpesvirus 4, Human; Heart Failure; Male; Female; Epstein-Barr Virus Infections; Middle Aged; Myocarditis; Aged; High-Throughput Nucleotide Sequencing; Myocardium; DNA, Viral; Adult; Biopsy
PubMed: 38892033
DOI: 10.3390/ijms25115845 -
International Journal of Molecular... May 2024Negeviruses are insect-specific enveloped RNA viruses that exhibit a wide geographic distribution. A novel nege-like virus, tentatively named nege-like virus (AGNLV,...
Negeviruses are insect-specific enveloped RNA viruses that exhibit a wide geographic distribution. A novel nege-like virus, tentatively named nege-like virus (AGNLV, GenBank: OR880429.1), was isolated from aphids () in Lijiang City, Yunnan, China. AGNLV has a genome sequence of 9258 nt (excluding the polyA tail) encoding three open reading frames (ORFs). ORF1 (7149 nt) encodes a viral methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase. ORF2 (1422 nt) encodes a DiSB-ORF2_chro domain and ORF3 encodes an SP24 domain. The genome sequence of AGNLV shares the highest nucleotide identity of 60.0% and 59.5% with Wuhan house centipede virus 1 (WHCV1) and Astegopteryx formosana nege-like virus (AFNLV), respectively. Phylogenetic analysis based on the RNA-dependent RNA polymerase shows that AGNLV is clustered with other negeviruses and nege-like viruses discovered in aphids, forming a distinct "unclassified clade". Interestingly, AGNLV only encodes three ORFs, whereas AFNLV and WHCV1 have four ORFs. Structure and transmembrane domain predictions show the presence of eight alpha helices and five transmembrane helices in the AGNLV ORF3. Translational enhancement of the AGNLV 5' UTR was similar to that of the 5' UTR of plant viruses. Our findings provide evidence of the diversity and structure of nege-like viruses and are the first record of such a virus from a member of the genus .
Topics: Animals; Aphids; Genome, Viral; Phylogeny; Open Reading Frames; China; RNA Viruses; RNA-Dependent RNA Polymerase; Viral Proteins; Insect Viruses; RNA, Viral
PubMed: 38891989
DOI: 10.3390/ijms25115802 -
International Journal of Molecular... May 2024Information generated via next-generation sequencing (NGS) technologies is often termed multi-omics data [...].
Information generated via next-generation sequencing (NGS) technologies is often termed multi-omics data [...].
Topics: Precision Medicine; Humans; Medical Oncology; High-Throughput Nucleotide Sequencing; Neoplasms; Data Science; Genomics
PubMed: 38891982
DOI: 10.3390/ijms25115797 -
International Journal of Molecular... May 2024The tyrosine kinase domain of the FMS-Like tyrosine kinase 3 (-TKD) is recurrently mutated in acute myeloid leukemia (AML). Common molecular techniques used in its...
The tyrosine kinase domain of the FMS-Like tyrosine kinase 3 (-TKD) is recurrently mutated in acute myeloid leukemia (AML). Common molecular techniques used in its detection include PCR and capillary electrophoresis, Sanger sequencing and next-generation sequencing with recognized sensitivity limitations. This study aims to validate the use of droplet digital PCR (ddPCR) in the detection of measurable residual disease (MRD) involving the common -TKD mutations (D835Y, D835H, D835V, D835E). Twenty-two diagnostic samples, six donor controls, and a commercial D835Y positive control were tested using a commercial Bio-rad ddPCR assay. All known variants were identified, and no false positives were detected in the wild-type control (100% specificity and sensitivity). The assays achieved a limit of detection suitable for MRD testing at 0.01% variant allelic fraction. Serial samples from seven intensively-treated patients with -TKD variants at diagnosis were tested. Five patients demonstrated clearance of -TKD clones, but two patients had -TKD persistence in the context of primary refractory disease. In conclusion, ddPCR is suitable for the detection and quantification of -TKD mutations in the MRD setting; however, the clinical significance and optimal management of MRD positivity require further exploration.
Topics: Humans; fms-Like Tyrosine Kinase 3; Neoplasm, Residual; Leukemia, Myeloid, Acute; Polymerase Chain Reaction; Mutation; Female; Male; Middle Aged; Aged; Adult; High-Throughput Nucleotide Sequencing
PubMed: 38891959
DOI: 10.3390/ijms25115771