Using next-generation sequencing of microRNAs to identify host and/or pathogen nucleic acid signatures in samples from children with biliary atresia – a pilot study

Biliary atresia (BA) is a progressive disease affecting infants resulting in inflammatory obliteration and fibrosis of the extra- and intra-hepatic biliary tree. BA may be grouped into type 1 isolated; type 2 syndromic, where other congenital malformations may be present; type 3 cystic BA, where there is cyst formation within an otherwise obliterated biliary tree; and cytomegalovirus-associated BA. The cause of BA is unclear, with immune dysregulation, inflammation and infection, particularly with cytomegalovirus (CMV), all implicated. In this study a total of 50/67 samples were tested for CMV DNA using quantitative real-time PCR. Ten liver tissue and 8 bile samples from 10 patients representing the range of BA types were also analysed by next-generation sequencing. CMV DNA was found in 8/50 (16 %) patients and a total of 265 differentially expressed microRNAs were identified. No statistically significant differences between the various types of BA were found. However, differences were identified in the expression patterns of 110 microRNAs in bile and liver tissue samples (P<0.05). A small number of bacterial and viral sequences were found, although their relevance to BA remains to be determined. No direct evidence of viral causes of BA were found, although clear evidence of microRNAs associated with hepatocyte and cholangiocyte differentiation together with fibrosis and inflammation were identified. These include miR-30 and the miR-23 cluster (liver and bile duct development) and miR-29, miR-483, miR-181, miR-199 and miR-200 (inflammation and fibrosis).

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A Novel Method for Microsatellite Instability Detection by Liquid Biopsy Based on Next- Generation Sequencing

Current Bioinformatics ◽

10.2174/1574893615666200324133451 ◽

2020 ◽

Vol 15 ◽

Author(s):

Zheng Jiang ◽

Hui Liu ◽

Siwen Zhang ◽

Jia Liu ◽

Weitao Wang ◽

...

Keyword(s):

Next Generation Sequencing ◽

Microsatellite Instability ◽

Liquid Biopsy ◽

Tumor Tissue ◽

High Sensitivity ◽

Next Generation ◽

Tissue Samples ◽

Next Generation Sequencing Technology ◽

Medication Selection ◽

Generation Sequencing

Background: Microsatellite instability (MSI) is a prognostic biomarker used to guide medication selection in multiple cancers, such as colorectal cancer. Traditional PCR with capillary electrophoresis and next-generation sequencing using paired tumor tissue and leukocyte samples are the main approaches for MSI detection due to their high sensitivity and specificity. Currently, patient tissue samples are obtained through puncture or surgery, which causes injury and risk of concurrent disease, further illustrating the need for MSI detection by liquid biopsy. Methods: We propose an analytic method using paired plasma/leukocyte samples and MSI detection using next-generation sequencing technology. Based on the theoretical progress of oncogenesis, we hypothesized that the microsatellite site length in plasma equals the combination of the distribution of tumor tissue and leukocytes. Thus, we defined a window-judgement method to identify whether biomarkers were stable. Results: Compared to traditional PCR as the standard, we evaluated three methods in 20 samples (MSI-H:3/MSS:17): peak shifting method using tissue vs. leukocytes, peak shifting method using plasma vs. leukocytes, and our method using plasma vs. leukocytes. Compared to traditional PCR, we observed a sensitivity of 100%, 0%, and 100%, and a specificity of 100.00%, 94.12%, and 88.24%, respectively. Conclusion: Our method has the advantage of possibly detecting MSI in a liquid biopsy and provides a novel direction for future studies to increase the specificity of the method.

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Panel of serum miRNAs as potential non-invasive biomarkers for pancreatic ductal adenocarcinoma

Scientific Reports ◽

10.1038/s41598-021-82266-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Imteyaz Ahmad Khan ◽

Safoora Rashid ◽

Nidhi Singh ◽

Sumaira Rashid ◽

Vishwajeet Singh ◽

...

Keyword(s):

Next Generation Sequencing ◽

Pancreatic Ductal Adenocarcinoma ◽

Early Stage ◽

Ductal Adenocarcinoma ◽

Next Generation ◽

Serum Samples ◽

Tissue Samples ◽

Non Invasive ◽

Specific Symptoms ◽

Generation Sequencing

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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Rapid Somatic Mutation Testing in Colorectal Cancer by Use of a Fully Automated System and Single-Use Cartridge: A Comparison with Next-Generation Sequencing

The Journal of Applied Laboratory Medicine ◽

10.1373/jalm.2018.026278 ◽

2018 ◽

Vol 3 (2) ◽

pp. 178-184 ◽

Cited By ~ 5

Author(s):

M Rabie Al-Turkmani ◽

Kelley N Godwin ◽

Jason D Peterson ◽

Gregory J Tsongalis

Keyword(s):

Colorectal Cancer ◽

Next Generation Sequencing ◽

Ffpe Tissue ◽

Braf V600e ◽

Automated System ◽

Next Generation ◽

Tissue Samples ◽

Tissue Sections ◽

Single Use ◽

Generation Sequencing

AbstractBackgroundMolecular tests have been increasingly used in the management of various cancers as more targeted therapies are becoming available as treatment options. The Idylla™ system is a fully integrated, cartridge-based platform that provides automated sample processing (deparaffinization, tissue digestion, and DNA extraction) and real-time PCR-based mutation detection with all reagents included in a single-use cartridge. This retrospective study aimed at evaluating both the Idylla KRAS and NRAS-BRAF-EGFR492 Mutation Assay cartridges (research use only) against next-generation sequencing (NGS) by using colorectal cancer (CRC) tissue samples.MethodsForty-four archived formalin-fixed paraffin-embedded (FFPE) CRC tissue samples previously analyzed by targeted NGS were tested on the Idylla system. Among these samples, 17 had a mutation in KRAS proto-oncogene, GTPase (KRAS), 5 in NRAS proto-oncogene, GTPase (NRAS), and 12 in B-Raf proto-oncogene, serine/threonine kinase (BRAF) as determined using the Ion AmpliSeq 50-gene Cancer Hotspot Panel v2. The remaining 10 samples were wild-type for KRAS, NRAS, and BRAF. Two 10-μm FFPE tissue sections were used for each Idylla run, 1 for the KRAS cartridge, and 1 for the NRAS-BRAF-EGFR492 cartridge. All cases met the Idylla minimum tumor content requirement for KRAS, NRAS, and BRAF (≥10%). Assay reproducibility was evaluated by testing commercial controls derived from human cell lines, which had an allelic frequency of 50% and were run in triplicate.ResultsThe Idylla system successfully detected all mutations previously identified by NGS in KRAS (G12C, G12D, G12V, G13D, Q61K, Q61R, A146T), NRAS (G12V, G13R, Q61H), and BRAF (V600E). Compared with NGS, Idylla had a sensitivity of 100%. Analysis of the mutated commercial controls demonstrated agreement with the expected result for all samples and 100% reproducibility. The Idylla system produced results quickly with a turnaround time of approximately 2 h.ConclusionThe Idylla system offers reliable and sensitive testing of clinically actionable mutations in KRAS, NRAS, and BRAF directly from FFPE tissue sections.

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Systematic Analysis of Transcriptomic Profile of Chondrocytes in Osteoarthritic Knee Using Next-Generation Sequencing and Bioinformatics

Journal of Clinical Medicine ◽

10.3390/jcm7120535 ◽

2018 ◽

Vol 7 (12) ◽

pp. 535 ◽

Cited By ~ 5

Author(s):

Yi-Jen Chen ◽

Wei-An Chang ◽

Ling-Yu Wu ◽

Ya-Ling Hsu ◽

Chia-Hsin Chen ◽

...

Keyword(s):

Next Generation Sequencing ◽

Family Member ◽

Subchondral Bone ◽

Joint Pain ◽

Expression Patterns ◽

Cartilage Damage ◽

Phenotypic Change ◽

Next Generation ◽

Arthritic Joint ◽

Generation Sequencing

The phenotypic change of chondrocytes and the interplay between cartilage and subchondral bone in osteoarthritis (OA) has received much attention. Structural changes with nerve ingrowth and vascular penetration within OA cartilage may contribute to arthritic joint pain. The aim of this study was to identify differentially expressed genes and potential miRNA regulations in OA knee chondrocytes through next-generation sequencing and bioinformatics analysis. Results suggested the involvement of SMAD family member 3 (SMAD3) and Wnt family member 5A (WNT5A) in the growth of blood vessels and cell aggregation, representing features of cartilage damage in OA. Additionally, 26 dysregulated genes with potential miRNA–mRNA interactions were identified in OA knee chondrocytes. Myristoylated alanine rich protein kinase C substrate (MARCKS), epiregulin (EREG), leucine rich repeat containing 15 (LRRC15), and phosphodiesterase 3A (PDE3A) expression patterns were similar among related OA cartilage, subchondral bone and synovial tissue arrays in Gene Expression Omnibus database. The Ingenuity Pathway Analysis identified MARCKS to be associated with the outgrowth of neurite, and novel miRNA regulations were proposed to play critical roles in the pathogenesis of the altered OA knee joint microenvironment. The current findings suggest new perspectives in studying novel genes potentially contributing to arthritic joint pain in knee OA, which may assist in finding new targets for OA treatment.

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No Metagenomic Evidence of Causative Viral Pathogens in Postencephalitic Parkinsonism Following Encephalitis Lethargica

Microorganisms ◽

10.3390/microorganisms9081716 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1716

Author(s):

Dániel Cadar ◽

Kurt A. Jellinger ◽

Peter Riederer ◽

Sabrina Strobel ◽

Camelia-Maria Monoranu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Neuronal Loss ◽

Unknown Etiology ◽

Next Generation ◽

Encephalitis Lethargica ◽

Tissue Samples ◽

Viral Pathogen ◽

Postencephalitic Parkinsonism ◽

Generation Sequencing ◽

Post Infectious

Postencephalitic parkinsonism (PEP) is a disease of unknown etiology and pathophysiology following encephalitis lethargica (EL), an acute-onset polioencephalitis of cryptic cause in the 1920s. PEP is a tauopathy with multisystem neuronal loss and gliosis, clinically characterized by bradykinesia, rigidity, rest tremor, and oculogyric crises. Though a viral cause of EL is likely, past polymerase chain reaction-based investigations in the etiology of both PEP and EL were negative. PEP might be caused directly by an unknown viral pathogen or the consequence of a post-infectious immunopathology. The development of metagenomic next-generation sequencing in conjunction with bioinformatic techniques has generated a broad-range tool for the detection of unknown pathogens in the recent past. Retrospective identification and characterization of pathogens responsible for past infectious diseases can be successfully performed with formalin-fixed paraffin-embedded (FFPE) tissue samples. In this study, we analyzed 24 FFPE brain samples from six patients with PEP by unbiased metagenomic next-generation sequencing. Our results show that no evidence for the presence of a specific or putative (novel) viral pathogen was found, suggesting a likely post-infectious immune-mediated etiology of PEP.

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Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events

Blood ◽

10.1182/blood-2010-12-324517 ◽

2011 ◽

Vol 118 (7) ◽

pp. 1903-1911 ◽

Cited By ~ 83

Author(s):

Luca Cecchetti ◽

Neal D. Tolley ◽

Noemi Michetti ◽

Loredana Bury ◽

Andrew S. Weyrich ◽

...

Keyword(s):

Next Generation Sequencing ◽

Matrix Metalloproteinases ◽

Mrna Expression ◽

Vascular Remodeling ◽

Differentially Express ◽

Expression Patterns ◽

Critical Role ◽

Rna Seq ◽

Next Generation ◽

Generation Sequencing

Abstract Megakaryocytes transfer a diverse and functional transcriptome to platelets during the final stages of thrombopoiesis. In platelets, these transcripts reflect the expression of their corresponding proteins and, in some cases, serve as a template for translation. It is not known, however, if megakaryocytes differentially sort mRNAs into platelets. Given their critical role in vascular remodeling and inflammation, we determined whether megakaryocytes selectively dispense transcripts for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) into platelets. Next-generation sequencing (RNA-Seq) revealed that megakaryocytes express mRNA for 10 of the 24 human MMP family members. mRNA for all of these MMPs are present in platelets with the exception of MMP-2, 14, and 15. Megakaryocytes and platelets also express mRNA for TIMPs 1-3, but not TIMP-4. mRNA expression patterns predicted the presence and, in most cases, the abundance of each corresponding protein. Nonetheless, exceptions were observed: MMP-2 protein is present in platelets but not its transcript. In contrast, quiescent platelets express TIMP-2 mRNA but only traces of TIMP-2 protein. In response to activating signals, however, platelets synthesize significant amounts of TIMP-2 protein. These results demonstrate that megakaryocytes differentially express mRNAs for MMPs and TIMPs and selectively transfer a subset of these into platelets. Among the platelet messages, TIMP-2 serves as a template for signal-dependent translation.

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Propionibacterium acnes: Disease-Causing Agent or Common Contaminant? Detection in Diverse Patient Samples by Next-Generation Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.02723-15 ◽

2016 ◽

Vol 54 (4) ◽

pp. 980-987 ◽

Cited By ~ 50

Author(s):

Sarah Mollerup ◽

Jens Friis-Nielsen ◽

Lasse Vinner ◽

Thomas Arn Hansen ◽

Stine Raith Richter ◽

...

Keyword(s):

Next Generation Sequencing ◽

Propionibacterium Acnes ◽

Opportunistic Pathogen ◽

Next Generation Sequencing Data ◽

Clinical Samples ◽

Next Generation ◽

Sequencing Data ◽

Tissue Samples ◽

Content Type ◽

Generation Sequencing

Propionibacterium acnesis the most abundant bacterium on human skin, particularly in sebaceous areas.P. acnesis suggested to be an opportunistic pathogen involved in the development of diverse medical conditions but is also a proven contaminant of human clinical samples and surgical wounds. Its significance as a pathogen is consequently a matter of debate. In the present study, we investigated the presence ofP. acnesDNA in 250 next-generation sequencing data sets generated from 180 samples of 20 different sample types, mostly of cancerous origin. The samples were subjected to either microbial enrichment, involving nuclease treatment to reduce the amount of host nucleic acids, or shotgun sequencing. We detected high proportions ofP. acnesDNA in enriched samples, particularly skin tissue-derived and other tissue samples, with the levels being higher in enriched samples than in shotgun-sequenced samples.P. acnesreads were detected in most samples analyzed, though the proportions in most shotgun-sequenced samples were low. Our results show thatP. acnescan be detected in practically all sample types when molecular methods, such as next-generation sequencing, are employed. The possibility of contamination from the patient or other sources, including laboratory reagents or environment, should therefore always be considered carefully whenP. acnesis detected in clinical samples. We advocate that detection ofP. acnesalways be accompanied by experiments validating the association between this bacterium and any clinical condition.

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Next Generation Sequencing Analysis of Biofilms from Three Dogs with Postoperative Surgical Site Infection

International Scholarly Research Notices ◽

10.1155/2014/282971 ◽

2014 ◽

Vol 2014 ◽

pp. 1-5 ◽

Cited By ~ 2

Author(s):

L. M. König ◽

R. Klopfleisch ◽

D. Höper ◽

A. D. Gruber

Keyword(s):

Next Generation Sequencing ◽

Sequencing Analysis ◽

Next Generation ◽

Bacterial Composition ◽

Tissue Samples ◽

Next Generation Sequencing Analysis ◽

Total Dna ◽

Chronic Wound Infections ◽

Generation Sequencing ◽

Formalin Fixed

The composition of biofilms in chronic wound infections of dogs is unclear. In the present study, histologically identified biofilms attached to sutures in chronically infected wounds of three dogs were examined by next generation sequencing of total DNA extracted from formalin-fixed and paraffin-embedded tissue samples. The analysis identified an inhomogeneous bacterial composition in three tissues containing biofilms. Some of the identified bacterial families such as Staphylococci and Streptococci have been found before in biofilms associated with human and canine wounds but in this study were quantitatively in the minority. The majority of the reads classified as bacterial sequences had the highest identity with sequences belonging to the Porphyromonadaceae, Deinococcaceae, Methylococcaceae, Nocardiaceae, Alteromonadaceae, and Propionibacteriaceae and thus taxons of so far minor relevance in veterinary medicine.

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Next-generation sequencing not superior to culture in periprosthetic joint infection diagnosis

The Bone & Joint Journal ◽

10.1302/0301-620x.103b1.bjj-2020-0017.r3 ◽

2021 ◽

Vol 103-B (1) ◽

pp. 26-31

Author(s):

Beau J. Kildow ◽

Sean P. Ryan ◽

Richard Danilkowicz ◽

Alexander L. Lazarides ◽

Colin Penrose ◽

...

Keyword(s):

Next Generation Sequencing ◽

Periprosthetic Joint Infection ◽

Joint Infection ◽

Next Generation ◽

Sample Collection ◽

Tissue Samples ◽

Musculoskeletal Infection ◽

Conventional Culture ◽

Significant Difference ◽

Generation Sequencing

Aims Use of molecular sequencing methods in periprosthetic joint infection (PJI) diagnosis and organism identification have gained popularity. Next-generation sequencing (NGS) is a potentially powerful tool that is now commercially available. The purpose of this study was to compare the diagnostic accuracy of NGS, polymerase chain reaction (PCR), conventional culture, the Musculoskeletal Infection Society (MSIS) criteria, and the recently proposed criteria by Parvizi et al in the diagnosis of PJI. Methods In this retrospective study, aspirates or tissue samples were collected in 30 revision and 86 primary arthroplasties for routine diagnostic investigation for PJI and sent to the laboratory for NGS and PCR. Concordance along with statistical differences between diagnostic studies were calculated. Results Using the MSIS criteria to diagnose PJI as the reference standard, the sensitivity and specificity of NGS were 60.9% and 89.9%, respectively, while culture resulted in sensitivity of 76.9% and specificity of 95.3%. PCR had a low sensitivity of 18.4%. There was no significant difference based on sample collection method (tissue swab or synovial fluid) (p = 0.760). There were 11 samples that were culture-positive and NGS-negative, of which eight met MSIS criteria for diagnosing infection. Conclusion In our series, NGS did not provide superior sensitivity or specificity results compared to culture. PCR has little utility as a standalone test for PJI diagnosis with a sensitivity of only 18.4%. Currently, several laboratory tests for PJI diagnosis should be obtained along with the overall clinical picture to help guide decision-making for PJI treatment. Cite this article: Bone Joint J 2021;103-B(1):26–31.

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