Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods

AbstractEarly-stage diagnosis of pancreatic ductal adenocarcinoma (PDAC) is difficult due to non-specific symptoms. Circulating miRNAs in body fluids have been emerging as potential non-invasive biomarkers for diagnosis of many cancers. Thus, this study aimed to assess a panel of miRNAs for their ability to differentiate PDAC from chronic pancreatitis (CP), a benign inflammatory condition of the pancreas. Next-generation sequencing was performed to identify miRNAs present in 60 FFPE tissue samples (27 PDAC, 23 CP and 10 normal pancreatic tissues). Four up-regulated miRNAs (miR-215-5p, miR-122-5p, miR-192-5p, and miR-181a-2-3p) and four down-regulated miRNAs (miR-30b-5p, miR-216b-5p, miR-320b, and miR-214-5p) in PDAC compared to CP were selected based on next-generation sequencing results. The levels of these 8 differentially expressed miRNAs were measured by qRT-PCR in 125 serum samples (50 PDAC, 50 CP, and 25 healthy controls (HC)). The results showed significant upregulation of miR-215-5p, miR-122-5p, and miR-192-5p in PDAC serum samples. In contrast, levels of miR-30b-5p and miR-320b were significantly lower in PDAC as compared to CP and HC. ROC analysis showed that these 5 miRNAs can distinguish PDAC from both CP and HC. Hence, this panel can serve as a non-invasive biomarker for the early detection of PDAC.

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Viral Surveillance in Serum Samples From Patients With Acute Liver Failure By Metagenomic Next-Generation Sequencing

Clinical Infectious Diseases ◽

10.1093/cid/cix596 ◽

2017 ◽

Vol 65 (9) ◽

pp. 1477-1485 ◽

Cited By ~ 42

Author(s):

Sneha Somasekar ◽

Deanna Lee ◽

Jody Rule ◽

Samia N Naccache ◽

Mars Stone ◽

...

Keyword(s):

Next Generation Sequencing ◽

Liver Failure ◽

Acute Liver Failure ◽

Next Generation ◽

Serum Samples ◽

Viral Surveillance ◽

Generation Sequencing

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MicroRNA exocytosis by large dense-core vesicle fusion

10.1101/093278 ◽

2016 ◽

Author(s):

Alican Gümürdü ◽

Ramazan Yildiz ◽

Erden Eren ◽

Gökhan Karakülah ◽

Turgay Ünver ◽

...

Keyword(s):

Next Generation Sequencing ◽

Vesicle Fusion ◽

Dense Core ◽

Snare Complex ◽

Dense Core Vesicle ◽

Next Generation ◽

Dense Core Vesicles ◽

Large Dense Core Vesicles ◽

Extracellular Mirna ◽

Generation Sequencing

AbstractNeurotransmitters and peptide hormones are secreted into outside the cell by a vesicle fusion process. Although non-coding RNA (ncRNA) that include microRNA (miRNA) regulates gene expression inside the cell where they are transcribed, extracellular miRNA has been recently discovered outside the cells, proposing that miRNA might be released to participate in cell-to-cell communication. Despite its importance of extracellular miRNA, the molecular mechanisms by which miRNA can be stored in vesicles and released by vesicle fusion remain enigmatic. Using next-generation sequencing, vesicle purification techniques, and synthetic neurotransmission, we observe that large dense-core vesicles (LDCVs) contain a variety of miRNAs including miR-375. Furthermore, miRNA exocytosis is mediated by the SNARE complex and accelerated by Ca2+. Our results suggest that miRNA can be a novel neuromodulator that can be stored in vesicles and released by vesicle fusion together with classical neurotransmitters.One Sentence SummaryUsing next-generation sequencing (NGS) for microRNA (miRNA) and synthetic neurotransmission, we observed that large dense-core vesicles (LDCVs) contain a variety of miRNA together with classical neurotransmitters, and that miRNA can be released by vesicle fusion mediated by SNARE.

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Next-Generation Sequencing to Detect Pathogens in Pediatric Febrile Neutropenia: A Single-Center Retrospective Study of 112 Cases

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab223 ◽

2021 ◽

Author(s):

Kazuhiro Horiba ◽

Yuka Torii ◽

Toshihiko Okumura ◽

Suguru Takeuchi ◽

Takako Suzuki ◽

...

Keyword(s):

Cluster Analysis ◽

Next Generation Sequencing ◽

Blood Culture ◽

Febrile Neutropenia ◽

Clinical Samples ◽

Next Generation ◽

Serum Samples ◽

Blood Samples ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Background Febrile neutropenia (FN) is a frequent complication in immunocompromised patients. However, causative microorganisms are detected in only 10% of patients. This study aimed to detect the microorganisms that cause FN using next-generation sequencing (NGS) to idenjpgy the genome derived from pathogenic microorganisms in the bloodstream. Here, we implemented a metagenomic approach to comprehensively analyze microorganisms present in clinical samples from patients with FN. Methods FN is defined as 1) a neutrophil count < 500/µL, and 2) fever ≥ 37.5 °C. Plasma/serum samples of 112 pediatric patients with FN, 10 patients with neutropenia without fever (NE), were sequenced by NGS and analyzed by a metagenomic pipeline PATHDET. Results The putative pathogens were detected by NGS in 5 of 10 patients with FN with positive for blood culture results, 15 of 87 patients (17%) with negative for blood culture results, and 3 of 8 patients with NE. Several bacteria that were common in the oral, skin, and gut flora were commonly detected in blood samples, suggesting translocation of the human microbiota to the bloodstream in the setting of neutropenia. The cluster analysis of the microbiota in blood samples using NGS demonstrated that the representative bacteria of each cluster was mostly consistent with the pathogens in each patient. Conclusions NGS technique has a great potential for detecting causative pathogens in patients with FN. Cluster analysis, which extracts characteristic microorganisms from a complex microbial population, may be effective to detect pathogens in minute quantities of microbiota, such as those from the bloodstream.

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Next-generation sequencing combined with serological tests based pathogen analysis for a neurocysticercosis patient with a 20-year history:a case report

BMC Neurology ◽

10.1186/s12883-021-02277-7 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Bin Chen ◽

Zheng Chen ◽

Yi-shu Yang ◽

Gui-lan Cai ◽

Xiao-jiao Xu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Taenia Solium ◽

Clinical Manifestations ◽

Next Generation ◽

Serum Samples ◽

Igg Antibody ◽

Serological Tests ◽

Diagnostic Efficacy ◽

Variable Sensitivity ◽

Generation Sequencing

Abstract Background Neurocysticercosis (NCC) is the most common helminthic infection of the central nervous system (CNS) caused by the larval stage of Taenia solium. Accurate and early diagnosis of NCC remains challenging due to its heterogeneous clinical manifestations, neuroimaging deficits, variable sensitivity, and specificity of serological tests. Next-generation sequencing (NGS)-based pathogen analysis in patient’s cerebrospinal fluid (CSF) with NCC infection has recently been reported indicating its diagnostic efficacy. In this case study, we report the diagnosis of a NCC patient with a symptomatic history of over 20 years using NGS analysis and further confirmation of the pathology by immunological tests. Case presentation This study reports the clinical imaging and immunological features of a patient with a recurrent headache for more than 20 years, which worsened gradually with the symptom of fever for more than 7 years and paroxysmal amaurosis for more than 1 year. By utilizing NGS technique, the pathogen was detected in patient’s CSF, and the presence of Taenia solium-DNA was confirmed by a positive immunological reaction to cysticercus IgG antibody in CSF and serum samples. The symptoms of the patient were alleviated, and the CSF condition was improved substantially after the anti-helminthic treatment. Conclusions This study suggests that combining CSF NGS with cysticercus IgG testing may be a highly promising approach for diagnosing the challenging cases of NCC. Further studies are needed to evaluate the parasitic DNA load in patients’ CSF for the diagnosis of disease severity, stage, and monitoring of therapeutic responses.

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Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing

RNA ◽

10.1261/rna.036863.112 ◽

2013 ◽

Vol 19 (5) ◽

pp. 712-722 ◽

Cited By ~ 118

Author(s):

K. L. Burgos ◽

A. Javaherian ◽

R. Bomprezzi ◽

L. Ghaffari ◽

S. Rhodes ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Next Generation Sequencing ◽

Next Generation ◽

Human Cerebrospinal Fluid ◽

Extracellular Mirna ◽

Generation Sequencing

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242. Comprehensive Pathogen Detection for Pediatric Febrile Neutropenia by Metagenomic Next-Generation Sequencing

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.317 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S137-S138

Author(s):

Kazuhiro Horiba ◽

Yuka Torii ◽

Yuichiro Hara ◽

Mayuko Shimada ◽

Takako Suzuki ◽

...

Keyword(s):

Next Generation Sequencing ◽

Blood Culture ◽

Febrile Neutropenia ◽

Diversity Index ◽

Clinical Sample ◽

Antimicrobial Treatment ◽

Microbial Pathogens ◽

Next Generation ◽

Serum Samples ◽

Generation Sequencing

Abstract Background Febrile neutropenia (FN) is a common complication in patients with solid tumors and hematologic malignancies. Identification of the causative microorganisms would contribute to optimization of antimicrobial treatment and thus improve the outcome of FN. However, causative microorganisms are detected in only 10% to 20% of FN patients. Next-generation sequencing (NGS) allows us to comprehensively analyze all microorganisms present in a clinical sample. In this study, we aimed to utilize NGS for the detection of microbial pathogens in infectious diseases and elucidate the infection source in FN. Methods FN is defined by two characteristics: (1) neutrophils count < 500/µL, and (2) fever ≥38.0°C. From 2016 to 2018, 112 plasma/serum samples of pediatric FN patients (11 positive blood cultures) were analyzed. Serum samples from 10 neutropenic patients without fever were also analyzed as controls. Shotgun sequencing method was applied for these samples. The metagenomic analyses were performed through the pipeline PATHDET, which has been newly established in our laboratory. Diagnosis based on NGS results was made based on the following criteria: (1) number of reads from all pathogens per million reads (PR) >650, (2) a specific pathogen’s reads per million reads (RPM) >200, and (3) diversity index >3.0. The NGS results were compared with those from blood culture. Results Sequencing reads of bacteria isolated through blood culture were identified by NGS in all 11 plasma/serum samples leading to the diagnosis of FN. The causative pathogens were diagnosed by NGS using the above criteria in 11 patients. However, the results were consistent with those of blood culture in only 4 samples. Of 101 cases with negative blood culture results, the causative pathogens were detected in 17 cases: Acinetobacter soli (2 cases), Burkholderia cepacian (1 case), Klebsiella variicola (1 case), and Roseomonas sp. (1 case) were identified at the species level. In addition, 7 cases (e.g., Acinetobacter) were identified at the genus level, and 5 cases (e.g., Enterobacteriaceae) were identified at the family level. Conclusion Metagenomic NGS technique has great potential for detecting causative pathogens with greater efficiency than the conventional methods. Disclosures All authors: No reported disclosures.

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Viral Concentration and Amplification from Human Serum Samples Prior to Application of Next-Generation Sequencing Analysis

The Human Virome - Methods in Molecular Biology ◽

10.1007/978-1-4939-8682-8_13 ◽

2018 ◽

pp. 173-188 ◽

Cited By ~ 3

Author(s):

Xavier Fernandez-Cassi ◽

Marta Rusiñol ◽

Sandra Martínez-Puchol

Keyword(s):

Next Generation Sequencing ◽

Human Serum ◽

Sequencing Analysis ◽

Next Generation ◽

Serum Samples ◽

Human Serum Samples ◽

Next Generation Sequencing Analysis ◽

Generation Sequencing

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Next-Generation Sequencing-Based Monitoring of Circulating Tumor DNA Reveals Clonotypic Heterogeneity in Untreated PTCL

Blood Advances ◽

10.1182/bloodadvances.2020003679 ◽

2021 ◽

Author(s):

Milos D Miljkovic ◽

Christopher Melani ◽

Stefania Pittaluga ◽

Rahul Lakhotia ◽

Andrea Nicole Lucas ◽

...

Keyword(s):

Next Generation Sequencing ◽

T Cell ◽

B Cell Lymphoma ◽

Circulating Tumor Dna ◽

Driver Mutations ◽

Next Generation ◽

Serum Samples ◽

Baseline Serum ◽

Tumor Dna ◽

Generation Sequencing

Peripheral T-cell lymphomas (PTCL) have marked biologic and clinical heterogeneity, which confounds treatment decisions. Advances in circulating tumor DNA (ctDNA) assays employing next generation sequencing (NGS) has improved the detection of molecular relapse and driver mutations in diffuse large B-cell lymphoma, and highlight the potential utility of ctDNA across lymphomas. We investigated NGS-based monitoring of T-cell receptor (TCR) sequences in PTCL patients undergoing frontline treatment (NCT00001337). Of 45 patients, 34 (76%) had tumor-specific clonotypes of the TCR β or ɣ genes identified, which included 18 (86%) from baseline tissue and 16 (67%) from baseline serum. Twenty-five (74%) patients had both TCRβ and TCRɣ clonotypes, 23 (68%) patients had more than one TCRɣ clonotype, and 4 (9%) had multiple TCRβ or TCRɣ clonotypes, demonstrating significant intra-patient clonotypic heterogeneity. Among 24 patients with available serial serum samples during treatment, 9 (38%) cleared ctDNA after 2 cycles of therapy, and 11 (46%) patients had detectable ctDNA at the end of treatment. Patients with detectable ctDNA after therapy showed a trend towards worse survival. Notably, two patients with persistently detectable ctDNA after therapy remain in remission with 10-years of follow-up. Clonotypic heterogeneity in tumors and persistence despite long-term remission suggests variability in oncological potential.

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Next-Generation Sequencing and Proteomics of Cerebrospinal Fluid From COVID-19 Patients With Neurological Manifestations

Frontiers in Immunology ◽

10.3389/fimmu.2021.782731 ◽

2021 ◽

Vol 12 ◽

Author(s):

Haijun Wang ◽

Zili Zhang ◽

Junfen Zhou ◽

Shuqing Han ◽

Zhenyu Kang ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Next Generation Sequencing ◽

Proteomic Analysis ◽

Case Series ◽

Cellular Interaction ◽

Neurological Manifestations ◽

Next Generation ◽

Serum Samples ◽

Generation Sequencing ◽

New Onset

The SARS-CoV-2 and its variants are still hitting the world. Ever since the outbreak, neurological involvements as headache, ageusia, and anosmia in COVID-19 patients have been emphasized and reported. But the pathogenesis of these new-onset neurological manifestations in COVID-19 patients is still obscure and controversial. As difficulty always lay in the diagnosis of neurological infection, current reports to validate the presence of SARS-CoV-2 in cerebrospinal fluid (CSF) almost relied on the basic methods and warranted improvement. Here we reported a case series of 8 patients with prominent new-onset neurological manifestations, who were screened out from a patch of 304 COVID-19 confirmed patients. Next-generation sequencing (NGS) and proteomics were conducted in the simultaneously obtained CSF and serum samples of the selected patients, with three non-COVID-19 patients with matched demographic features used as the controls for proteomic analysis. SARS-CoV-2 RNA was detected in the CSF of four COVID-19 patients and was suspicious in the rest four remaining patients by NGS, but was negative in all serum samples. Proteomic analysis revealed that 185 and 59 proteins were differentially expressed in CSF and serum samples, respectively, and that only 20 proteins were shared, indicating that the proteomic changes in CSF were highly specific. Further proteomic annotation highlighted the involvement of complement system, PI3K-Akt signaling pathway, enhanced cellular interaction, and macrophages in the CSF proteomic alterations. This study, equipped with NGS and proteomics, reported a high detection rate of SARS-CoV-2 in the CSF of COVID-19 patients and the proteomic alteration of CSF, which would provide insights into understanding the pathological mechanism of SARS-CoV-2 CNS infection.

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