Next-generation sequencing predicts interaction network between miRNA and target genes in lipoteichoic acid-stimulated human neutrophils

Citrus dwarfing viroid (CDVd) induces stunting on sweet orange trees [Citrus sinensis (L.) Osbeck], propagated on trifoliate orange rootstock [Citrus trifoliata (L.), syn. Poncirus trifoliata (L.) Raf.]. MicroRNAs (miRNAs) are a class of non-coding small RNAs (sRNAs) that play important roles in the regulation of tree gene expression. To identify miRNAs in dwarfed citrus trees, grown in high-density plantings, and their response to CDVd infection, sRNA next-generation sequencing was performed on CDVd-infected and non-infected controls. A total of 1,290 and 628 miRNAs were identified in stem and root tissues, respectively, and among those, 60 were conserved in each of these two tissue types. Three conserved miRNAs (csi-miR479, csi-miR171b, and csi-miR156) were significantly downregulated (adjusted p-value < 0.05) in the stems of CDVd-infected trees compared to the non-infected controls. The three stem downregulated miRNAs are known to be involved in various physiological and developmental processes some of which may be related to the characteristic dwarfed phenotype displayed by CDVd-infected C. sinensis on C. trifoliata rootstock field trees. Only one miRNA (csi-miR535) was significantly downregulated in CDVd-infected roots and it was predicted to target genes controlling a wide range of cellular functions. Reverse transcription quantitative polymerase chain reaction analysis performed on selected miRNA targets validated the negative correlation between the expression levels of these targets and their corresponding miRNAs in CDVd-infected trees. Our results indicate that CDVd-responsive plant miRNAs play a role in regulating important citrus growth and developmental processes that may participate in the cellular changes leading to the observed citrus dwarf phenotype.

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Exosomal miRNAs are potential diagnostic biomarkers between malignant and benign thyroid nodules based on next-generation sequencing

Carcinogenesis ◽

10.1093/carcin/bgz160 ◽

2019 ◽

Cited By ~ 2

Author(s):

Qunxiong Pan ◽

Jiangman Zhao ◽

Mingzhu Li ◽

Xiaoyu Liu ◽

Yaping Xu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Thyroid Nodule ◽

Target Genes ◽

Nodular Goiter ◽

Needle Aspiration ◽

Mitogen Activated Protein Kinase ◽

Small Rna Sequencing ◽

Next Generation ◽

Micro Rnas ◽

Generation Sequencing

Abstract An accurate biomarker or method for diagnosis of thyroid nodule with indeterminate fine-needle aspiration result is essential for clinical treatment. Micro RNAs (miRNAs) of exosomes are advantageous in the diagnosis of tumors because they are highly stable, and be protected by a bilayer membrane structure. Exosomes were isolated from 13 papillary thyroid carcinoma (PTC) and 7 nodular goiter (NG) patients’ plasma. Small RNA sequencing was performed on exosomes’ RNA in next-generation sequencing (NGS) platform. Then, we performed comprehensive analysis on miRNA expression profile in exosome of two groups. One hundred and twenty-nine differentially expressed miRNAs were identified in plasma exosomes between PTC and NG patients. Forty-nine miRNAs were up-regulated, and 80 miRNAs were down-regulated in PTC patients. Receiver operating characteristic (ROC) curves of 129 miRNAs were plotted. Area under curve (AUC) of 129 miRNAs was 0.571–0.951, with distribution peak of 0.82–0.86. AUC of 11 miRNAs was above 0.9, miR-5189-3p had the most optimal performance for diagnosis between PTC and NG, with 0.951 of AUC. Target genes of 129 miRNAs were enriched into 7 cancer-related signaling pathways, including mitogen-activated protein kinase (MAPK), tumor necrosis factor (TNF), NF-kappa B signaling pathway and so on. This study profiled the miRNA signature of exosomes from PTC patients and NG patients. We proposed a group of miRNAs in plasma exosomes as candidate biomarkers for thyroid nodule diagnosis.

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Next generation sequencing allows deeper analysis and understanding of genomes and transcriptomes including aspects to fertility

Reproduction Fertility and Development ◽

10.1071/rd10247 ◽

2011 ◽

Vol 23 (1) ◽

pp. 75 ◽

Cited By ~ 7

Author(s):

Thomas Werner

Keyword(s):

Next Generation Sequencing ◽

Transcriptional Control ◽

Target Genes ◽

De Novo ◽

Alternative Promoters ◽

Next Generation ◽

Sequencing Data ◽

Genome Wide ◽

A Genome ◽

Generation Sequencing

Reproduction and fertility are controlled by specific events naturally linked to oocytes, testes and early embryonal tissues. A significant part of these events involves gene expression, especially transcriptional control and alternative transcription (alternative promoters and alternative splicing). While methods to analyse such events for carefully predetermined target genes are well established, until recently no methodology existed to extend such analyses into a genome-wide de novo discovery process. With the arrival of next generation sequencing (NGS) it becomes possible to attempt genome-wide discovery in genomic sequences as well as whole transcriptomes at a single nucleotide level. This does not only allow identification of the primary changes (e.g. alternative transcripts) but also helps to elucidate the regulatory context that leads to the induction of transcriptional changes. This review discusses the basics of the new technological and scientific concepts arising from NGS, prominent differences from microarray-based approaches and several aspects of its application to reproduction and fertility research. These concepts will then be illustrated in an application example of NGS sequencing data analysis involving postimplantation endometrium tissue from cows.

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Analytical performance of a highly sensitive system to detect gene variants using next-generation sequencing for lung cancer companion diagnostics

10.1101/2021.10.13.21264976 ◽

2021 ◽

Author(s):

Kikuya Kato ◽

Jiro Okami ◽

Harumi Nakamura ◽

Keiichiro Honma ◽

Yoshiharu Sato ◽

...

Keyword(s):

Next Generation Sequencing ◽

Target Genes ◽

Analytical Performance ◽

Braf V600e ◽

Targeted Agents ◽

Next Generation ◽

Companion Diagnostics ◽

Cell Lung Carcinoma ◽

Molecular Targeted Agents ◽

Generation Sequencing

Companion diagnostics, which predict the efficacy of molecular targeted agents based on genetic information, are indispensable for the treatment of advanced non-small cell lung carcinoma. Recent increase in the number of molecular targeted agents and the corresponding target genes have led to the demand for the simultaneous testing of multiple genes. Although gene panels using next-generation sequencing (NGS panels) are ideal for this purpose, conventional panels require high tumor content, and biopsy samples often do not meet this requirement. We developed a new NGS panel called a compact panel, to accommodate biopsy samples without the restriction of tumor content. The compact panel is characterized by high sensitivity, with detection limits for mutations of 0.14%, 0.20%, 0.48%, 0.24%, and 0.20% for EGFR exon 19 deletion, L858R, T790M, BRAF V600E, and KRAS G12C, respectively. Mutation detection also has a high quantitative ability, with correlation coefficients ranging from 0.966 to 0.992. The panel detected fusions in samples whose tumor cell content was as low as 1%. The compact panel exhibited good concordance with approved tests as follows: EGFR positive, 100.0 (95% confidence interval 95.5-100); EGFR negative, 90.9 (82.2-96.3); ALK positive, 96.7 (83.8-99.9); ALK negative, 98.4 (97.2-99.2); ROS1 positive, 100 (66.4-100); ROS1 negative, 99.0 (97.1-99.2); MET positive, 98.0 (89.0-100); MET negative 100 (92.8-100). The analytical performance demonstrated that the compact panel can handle various types of biopsy samples obtained by routine clinical practice, without requiring strict pathological monitoring like in case of conventional NGS panels.

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Target Capture/Next-Generation Sequencing for Nonsyndromic Cleft Lip and Palate in the Japanese Population

The Cleft Palate-Craniofacial Journal ◽

10.1177/1055665619857650 ◽

2019 ◽

Vol 57 (1) ◽

pp. 80-87

Author(s):

Masayasu Shibano ◽

Akira Watanabe ◽

Nobuo Takano ◽

Hiroyuki Mishima ◽

Akira Kinoshita ◽

...

Keyword(s):

Next Generation Sequencing ◽

Cleft Palate ◽

Mutation Analysis ◽

Japanese Population ◽

Cleft Lip ◽

Target Genes ◽

De Novo Mutation ◽

Genome Wide Association Studies ◽

Next Generation ◽

Generation Sequencing

Objective: The pathogenesis of nonsyndromic cleft lip with or without cleft palate (NSCL ± P) and nonsyndromic cleft palate only (NSCP) may be associated with genetic factors. Although some predisposing genes/loci have been reported, their attributable risk is too small to be clinically meaningful. To clarify the genetic causes and mechanisms of NSCL±P or NSCP, we conducted mutation analysis of target genes using a next-generation sequencing (NGS) approach. Methods: The target genes, IRF6, WNT5A, WNT9B, TP63, MSX1, TFAP2A, PAX9, DLX3, DLX4, and MN1, were selected based on previous reports of potential associations with the development of NSCL±P or NSCP from genome-wide association studies and candidate gene analyses. Mutation analysis was conducted using NGS on 74 Japanese trios (patient and parents) and 18 Japanese patients only families. Results: We detected single-nucleotide variants (SNVs) for 7 genes: IRF6, DLX4, WNT5A, TFAP2A, WNT9B, TP63, and PAX9. The SNVs found on IRF6 and DLX4 were missense mutations, whereas those identified on WNT5A, TFAP2A, WNT9B, TP63, and PAX9 were rare variants in the noncoding region; no de novo mutation was identified in the trio samples. The amino acid change on DLX4 was detected within the highly conserved homeodomain and was predicted to have a deleterious impact on the protein function by in silico analysis. Conclusions: The DLX4 missense mutation c.359C>T (Pro120Leu) was found in 1 Japanese patient with NSCL±P and was located in the homeodomain region. This mutation likely plays a role in the development of NSCL±P in the Japanese population.

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Next generation sequencing based detection of 15 target genes mutations in papillary thyroid carcinoma

Precision Medical Sciences ◽

10.1002/prm2.12028 ◽

2020 ◽

Vol 9 (2) ◽

pp. 90-93

Author(s):

Zhuo Wang ◽

Changwen Jing ◽

Haixia Cao ◽

SiWen Liu ◽

Jianzhong Wu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Papillary Thyroid Carcinoma ◽

Thyroid Carcinoma ◽

Target Genes ◽

Papillary Thyroid ◽

Next Generation ◽

Generation Sequencing

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PPARG: Gene Expression Regulation and Next-Generation Sequencing for Unsolved Issues

PPAR Research ◽

10.1155/2010/409168 ◽

2010 ◽

Vol 2010 ◽

pp. 1-17 ◽

Cited By ~ 34

Author(s):

Valerio Costa ◽

Maria Assunta Gallo ◽

Francesca Letizia ◽

Marianna Aprile ◽

Amelia Casamassimi ◽

...

Keyword(s):

Next Generation Sequencing ◽

Conformational Changes ◽

Target Genes ◽

Gene Expression Regulation ◽

Massively Parallel Sequencing ◽

Protein Isoforms ◽

Peroxisome Proliferator ◽

Next Generation ◽

The Impact ◽

Generation Sequencing

Peroxisome proliferator-activated receptor gamma (PPARγ) is one of the most extensively studied ligand-inducible transcription factors (TFs), able to modulate its transcriptional activity through conformational changes. It is of particular interest because of its pleiotropic functions: it plays a crucial role in the expression of key genes involved in adipogenesis, lipid and glucid metabolism, atherosclerosis, inflammation, and cancer. Its protein isoforms, the wide number of PPARγtarget genes, ligands, and coregulators contribute to determine the complexity of its function. In addition, the presence of genetic variants is likely to affect expression levels of target genes although the impact ofPPARGgene variations on the expression of target genes is not fully understood. The introduction of massively parallel sequencing platforms—in the Next Generation Sequencing (NGS) era—has revolutionized the way of investigating the genetic causes of inherited diseases. In this context, DNA-Seq for identifying—within both coding and regulatory regions ofPPARGgene—novel nucleotide variations and haplotypes associated to human diseases, ChIP-Seq for defining a PPARγbinding map, and RNA-Seq for unraveling the wide and intricate gene pathways regulated by PPARG, represent incredible steps toward the understanding of PPARγin health and disease.

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Alterations in Esophageal MicroRNAs Expression in Primary Esophageal Achalasia by Next-Generation Sequencing

10.21203/rs.2.24777/v2 ◽

2020 ◽

Author(s):

Mahin Gholipour ◽

Javad Mikaeli ◽

Seyed Javad Mowla ◽

Mohammad Reza Bakhtiarizadeh ◽

Marie Saghaeian Jazi ◽

...

Keyword(s):

Next Generation Sequencing ◽

Mirna Expression ◽

Target Genes ◽

Neuronal Cell ◽

Esophageal Achalasia ◽

P Value ◽

Next Generation ◽

Candidate Mirnas ◽

Generation Sequencing

Abstract Background: Molecular knowledge regarding the primary esophageal achalasia is essential for early diagnosis and treatment of this neurodegenerative motility disorder. So, there is a need to find the main microRNAs (miRNAs) contributing to the mechanisms of achalasia.Methods: This study was conducted to determine some patterns of deregulated miRNAs in the achalasia. This case-control study was performed on 52 patients with achalasia and 50 non-achalasia controls. The miRNA expression profiling was conducted on the esophageal tissue samples obtained from the patients with achalasia and non-achalasia patients controls using the Next-Generation Sequencing (NGS). Differential expression of miRNAs was analyzed by the edgeR software. The selected dysregulated miRNAs were additionally confirmed using the quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Potential target genes of the downregulated and upregulated miRNAs were also predicted to understand the putative role of the miRNAs in the development of the achalasia. Results: Totally, 15 miRNAs were identified that were significantly altered in the tissues of the patients with achalasia compared to the controls. Among them, three miRNAs including miR-133a-5p, miR-143-3p, and miR-6507-5p were upregulated. Also, six miRNAs including miR-215-5p, miR-216a-5p, miR-216b-5p, miR-217, miR-7641, and miR-194-5p were downregulated significantly. The predicted targets for the dysregulated miRNAs showed significant disease-associated pathways like neuronal cell apoptosis, neuromuscular balance, nerve growth factor signaling, and immune response regulation. Gene expression analysis confirmed significant downregulation of the hsa-miR-217 (p-value = 0.004) in the Lower Esophageal Sphincter (LES) of the patients with achalasia with significant enrichment in the myelination process ontology. The results of this study provide the first integrated miRNA expression profile in the achalasia using the NGS. Our findings introduced 15 candidate miRNAs as achalasia-associated non-coding RNAs and also confirmed the downregulation of the hsa-miR-217 in the achalasia disease. Conclusions: Our results may serve as a basis for more future functional studies to investigate the role of candidate miRNAs in the etiology of achalasia and their application in diagnosis and probably treatment of the disease.

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Alterations in esophageal microRNAs expression in primary esophageal achalasia by next-generation sequencing

10.21203/rs.2.24777/v1 ◽

2020 ◽

Author(s):

Mahin Gholipour ◽

Javad Mikaeli ◽

Seyed Javad Mowla ◽

Mohammad Reza Bakhtiarizadeh ◽

Marie Saghaeian Jazi ◽

...

Keyword(s):

Next Generation Sequencing ◽

Mirna Expression ◽

Target Genes ◽

Esophageal Achalasia ◽

P Value ◽

Growth Factor Signaling ◽

Next Generation ◽

Tissue Samples ◽

Generation Sequencing

Abstract Background The molecular knowledge of primary esophageal achalasia is essential for the early diagnosis and treatment of this neurodegenerative motility disorder. So it is substantial to find the main microRNAs (miRNAs), which are related to mechanisms of achalasia. Methods This study aimed to determine some patterns of deregulated miRNAs in achalasia. This case-control study was performed on 52 achalasia patients and 50 non-achalasia controls. The miRNA expression profiling was conducted on esophageal tissue samples from achalasia and non-achalasia patients, via next-generation sequencing (NGS). Differential expression of miRNAs was analyzed by edgeR software. The selected dysregulated miRNAs were additionally confirmed using RT-qPCR. Potential target genes of the down-regulated and up-regulated miRNAs were also predicted to understand the putative role of the miRNAs in achalasia. Results We identified 15 miRNAs that were significantly altered in the achalasia tissues compared to controls. Among these miRNAs, three; miR-133a-5p, miR-143-3p and miR-6507-5p were up-regulated. Also we found six miRNAs; miR-215-5p, miR-216a-5p, miR-216b-5p, miR-217, miR-7641 and miR-194-5p were down-regulated significantly. The predicted targets for the dysregulated miRNAs showed significant disease-associated pathways like neuron cell apoptosis, neuromuscular balance, neuron growth factor signaling and immune response regulation. Gene expression analysis confirmed significant downregulation of hsa-miR-217 (p-value =0.004) in LES of achalasia patients with significant enrichment in myelination process ontology. This study provides the first integrated miRNA expression profile using NGS in achalasia. Our findings introduced 15 candidate microRNAs as achalasia associated non-coding RNAs genes showing confirmed downregulation of the hsa-miR-217 in Achalasia disease. Conclusions Our results may serve as a basis for more future functional studies to discover the role of candidate miRNAs in the etiology of achalasia and their application in diagnosis and probably treatment.

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Genotyping of 25 Leukemia-Associated Genes in a Single Work Flow by Next-Generation Sequencing Technology with Low Amounts of Input Template DNA

Clinical Chemistry ◽

10.1373/clinchem.2013.204099 ◽

2013 ◽

Vol 59 (8) ◽

pp. 1238-1250 ◽

Cited By ~ 14

Author(s):

Jenny Rinke ◽

Vivien Schäfer ◽

Mathias Schmidt ◽

Janine Ziermann ◽

Alexander Kohlmann ◽

...

Keyword(s):

Next Generation Sequencing ◽

Target Genes ◽

Work Flow ◽

Next Generation ◽

Next Generation Sequencing Technology ◽

Wide Range ◽

Next Generation Sequencing Ngs ◽

Template Dna ◽

Ngs Data ◽

Generation Sequencing

BACKGROUND We sought to establish a convenient, sensitive next-generation sequencing (NGS) method for genotyping the 26 most commonly mutated leukemia-associated genes in a single work flow and to optimize this method for low amounts of input template DNA. METHODS We designed 184 PCR amplicons that cover all of the candidate genes. NGS was performed with genomic DNA (gDNA) from a cohort of 10 individuals with chronic myelomonocytic leukemia. The results were compared with NGS data obtained from sequencing of DNA generated by whole-genome amplification (WGA) of 20 ng template gDNA. Differences between gDNA and WGA samples in variant frequencies were determined for 2 different WGA kits. RESULTS For gDNA samples, 25 of 26 genes were successfully sequenced with a sensitivity of 5%, which was achieved by a median coverage of 492 reads (range, 308–636 reads) per amplicon. We identified 24 distinct mutations in 11 genes. With WGA samples, we reliably detected all mutations above 5% sensitivity with a median coverage of 506 reads (range, 256–653 reads) per amplicon. With all variants included in the analysis, WGA amplification by the 2 kits tested yielded differences in variant frequencies that ranged from −28.19% to +9.94% [mean (SD) difference, −0.2% (4.08%)] and from −35.03% to +18.67% [mean difference, −0.75% (5.12%)]. CONCLUSIONS Our method permits simultaneous analysis of a wide range of leukemia-associated target genes in a single sequencing run. NGS can be performed after WGA of template DNA for reliable detection of variants without introducing appreciable bias.

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