scholarly journals Development and Optimization of Metagenomic Next-Generation Sequencing Methods for Cerebrospinal Fluid Diagnostics

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Patricia J. Simner ◽  
Heather B. Miller ◽  
Florian P. Breitwieser ◽  
Gabriel Pinilla Monsalve ◽  
Carlos A. Pardo ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Sample Pretreatment ◽  
Standard Of Care ◽  
Nucleic Acid Amplification ◽  
Next Generation ◽  
Bead Beating ◽  
Dna And Rna ◽  
Positive Threshold ◽  
Generation Sequencing

ABSTRACT The purpose of this study was to develop and optimize different processing, extraction, amplification, and sequencing methods for metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) specimens. We applied mNGS to 10 CSF samples with known standard-of-care testing (SoC) results (8 positive and 2 negative). Each sample was subjected to nine different methods by varying the sample processing protocols (supernatant, pellet, neat CSF), sample pretreatment (with or without bead beating), and the requirement of nucleic acid amplification steps using DNA sequencing (DNASeq) (with or without whole-genome amplification [WGA]) and RNA sequencing (RNASeq) methods. Negative extraction controls (NECs) were used for each method variation (4/CSF sample). Host depletion (HD) was performed on a subset of samples. We correctly determined the pathogen in 7 of 8 positive samples by mNGS compared to SoC. The two negative samples were correctly interpreted as negative. The processing protocol applied to neat CSF specimens was found to be the most successful technique for all pathogen types. While bead beating introduced bias, we found it increased the detection yield of certain organism groups. WGA prior to DNASeq was beneficial for defining pathogens at the positive threshold, and a combined DNA and RNA approach yielded results with a higher confidence when detected by both methods. HD was required for detection of a low-level-positive enterovirus sample. We demonstrate that NECs are required for interpretation of these complex results and that it is important to understand the common contaminants introduced during mNGS. Optimizing mNGS requires the use of a combination of techniques to achieve the most sensitive, agnostic approach that nonetheless may be less sensitive than SoC tools.

The applications of next-generation sequencing (NGS) in drug development for cancer therapy

2020 ◽  
Vol 16 ◽  
Author(s):  
Pelin Telkoparan-Akillilar ◽  
Dilek Cevik
Keyword(s):  
Next Generation Sequencing ◽  
Drug Discovery ◽  
Cancer Therapy ◽  
Target Identification ◽  
Rapid Development ◽  
Next Generation ◽  
Biomedical Sciences ◽  
Dna And Rna ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Background: Numerous sequencing techniques have been progressed since the 1960s with the rapid development of molecular biology studies focusing on DNA and RNA. Methods: a great number of articles, book chapters, websites are reviewed, and the studies covering NGS history, technology and applications to cancer therapy are included in the present article. Results: High throughput next-generation sequencing (NGS) technologies offer many advantages over classical Sanger sequencing with decreasing cost per base and increasing sequencing efficiency. NGS technologies are combined with bioinformatics software to sequence genomes to be used in diagnostics, transcriptomics, epidemiologic and clinical trials in biomedical sciences. The NGS technology has also been successfully used in drug discovery for the treatment of different cancer types. Conclusion: This review focuses on current and potential applications of NGS in various stages of drug discovery process, from target identification through to personalized medicine.

scholarly journals Clinical diagnostic application of metagenomic next-generation sequencing in children with severe pneumonia

2019 ◽  
Author(s):  
Heping Wang ◽  
Zhiwei Lu ◽  
Yaomin Bao ◽  
Yonghong Yang ◽  
Ronald de Groot ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Mycoplasma Pneumoniae ◽  
Severe Pneumonia ◽  
Clinical Diagnostics ◽  
Nucleic Acid Amplification ◽  
Direct Immunofluorescence ◽  
Specific Information ◽  
Next Generation ◽  
Blood Tests ◽  
Generation Sequencing

Abstract Background: Pneumonia is one of the most important causes of morbidity and mortality in children. Identification and characterization of pathogens that cause infections are crucial for accurate treatment and accelerated recovery of the patients. However, in most cases the causative agent cannot be identified partly due to the limited spectrum covered by current diagnostics based on nucleic acid amplification. Therefore, in this study we explored the application of metagenomic next-generation sequencing (mNGS) for the diagnosis of children with severe pneumonia. Methods: From April to July 2017, 32 children were hospitalized with severe pneumonia in Shenzhen Children’s Hospital. Blood tests were conducted immediately after hospitalization to assess infection, oropharygeal swabs were collected to identify common pathogens. After bronchoscopy, bronchoalveolar lavage fluids (BALFs) were collected for further pathogen identification using standardized laboratory and mNGS. Results: Blood tests were normal in 3 of the 32 children. In oropharygeal swabs from 5 patients Mycoplasma pneumoniae by qPCR, 27 cases showed negative results for common pathogens. In BALFs we detected 6 cases with Mycoplasma pneumoniae with qPCR, 9 patients with adenovirus by using a Direct Immunofluorescence Assay (DFA) and 4 patients with bacterial infections, as determined by culture, In 3 of the cases a co-infection was detected. In 15 cases no common pathogens were found in BALF samples, using the current diagnostics, while in all the 32 BALFS pathogens were identified using mNGS, including adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, cytomegalovirus andbocavirus. Conclusions: mNGS can increase the sensitivity of detection of the causative pathogens in children with severe pneumonia. In addition, mNGS will give more strain specific information, will help to identify new pathogens and could potentially help to trace and control outbreaks. In this study we have shown that it is feasible to have the results within 24 hours, making the application of mNGS feasible for clinical diagnostics.

scholarly journals Investigation of methods for extraction of bacterial DNA metagenome from goat rumen

2018 ◽  
Vol 15 (2) ◽  
pp. 367-372
Author(s):  
Lê Ngọc Giang ◽  
Lưu Hàn Ly ◽  
Nguyễn Mai Phương ◽  
Lê Tùng Lâm ◽  
Đỗ Thị Huyền ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rumen Fluid ◽  
Next Generation ◽  
Taq Polymerase ◽  
Bead Beating ◽  
Sequencing Technologies ◽  
Polymerase Inhibitor ◽  
Stool Dna ◽  
Generation Sequencing ◽  
Goat Rumen

Microorganisms, particularly bacteria, in the ruminant's rumen are valuable genetic resources that many scientists interested in. In recent years, the application of next-generation sequencing technologies allows direct decoding an extracted DNA metagenome in each ecological community without culture, increasing the efficiency of exploiting interested genes. Notably, the quantity and quality of extracted DNA play an important role in getting a reliable metagenome database. In this study, DNA metagenome from goat rumen fluid was extracted by five different methods RBB (repeated bead beating plus column), RBBC (repeated bead beating), PSP1, PSP2 (PSP®Spin Stool DNA Kit, protocol 1, 2, Germany) và QIA (QIAamp® DNA Stool Mini Kit, Germany). The results showed that DNA metagenome obtained by all methods had A260/280 greater than 1.8. DNA extracted by the RBB method had high DNA concentration but low A260/230 values (less than 1.4) and still contained Taq polymerase inhibitor. After purifying by QIA column, A260/230 values of RBB-extracted DNA significantly increased up to 2.0 and Taq polymerase inhibitor in samples were removed. However, the concentrations decreased by 57% that nearly equivalent to concentration of DNA metagenome obtained by QIA. The method using PSP®Spin Stool DNA kit produced the highest DNA concentrations (from 149.7 to 195.5 ng/µl) with A260/280 ratios of 1.9 and A260/230 ratios of 1.8 to 1.9. Morever, this method was able to remove polymerase inhibitor and be performed on short time. Therefore, the PSP®Spin Stool DNA kit is a suitable method for DNA metagenome extraction of bacteria from goat rumen. DNA obtained by this method fulfilled all criteria about quality and concentration for sequencing by next-generation sequencing Illumina.

scholarly journals Diagnosis of Enterococcus faecalis meningitis associated with long-term cerebrospinal fluid rhinorrhoea using metagenomics next-generation sequencing: a case report

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xiaobo Zhang ◽  
Chao Jiang ◽  
Chaojun Zhou
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Enterococcus Faecalis ◽  
Bone Defect ◽  
Next Generation ◽  
History Of ◽  
Diagnostic Approaches ◽  
Post Traumatic ◽  
Skull Bone Defect ◽  
Generation Sequencing

Abstract Background Enterococcus faecalis (E. faecalis) meningitis is a rare disease, and most of its occurrences are of post-operative origin. Its rapid diagnosis is critical for effective clinical management. Currently, the diagnosis is focused on cerebrospinal fluid (CSF) culture, but this is quite limited. By comparison, metagenomic next-generation sequencing (mNGS) can overcome the deficiencies of conventional diagnostic approaches. To our knowledge, mNGS analysis of the CSF in the diagnosis of E. faecalis meningitis has been not reported. Case presentation We report the case of E. faecalis meningitis in a 70-year-old female patient without a preceding history of head injury or surgery, but with an occult sphenoid sinus bone defect. Enterococcus faecalis meningitis was diagnosed using mNGS of CSF, and she recovered satisfactorily following treatment with appropriate antibiotics and surgical repair of the skull bone defect. Conclusions Non-post-traumatic or post-surgical E. faecalis meningitis can occur in the presence of occult defects in the cranium, and mNGS technology could be helpful in diagnosis in the absence of a positive CSF culture.

scholarly journals Metagenomic Next-Generation Sequencing of Cerebrospinal Fluid for the Diagnosis of External Ventricular and Lumbar Drainage-Associated Ventriculitis and Meningitis

2020 ◽  
Vol 11 ◽  
Author(s):  
Lingye Qian ◽  
Yijun Shi ◽  
Fangqiang Li ◽  
Yufei Wang ◽  
Miao Ma ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Staphylococcus Epidermidis ◽  
Coincidence Rate ◽  
Next Generation ◽  
Lumbar Drainage ◽  
Gram Positive Bacteria ◽  
Neurosurgical Patients ◽  
Generation Sequencing ◽  
Guided Therapy

Metagenomic next-generation sequencing (mNGS) has become a widely used technology that can accurately detect individual pathogens. This prospective study was performed between February 2019 and September 2019 in one of the largest clinical neurosurgery centers in China. The study aimed to evaluate the performance of mNGS on cerebrospinal fluid (CSF) from neurosurgical patients for the diagnosis of external ventricular and lumbar drainage (EVD/LD)-associated ventriculitis and meningitis (VM). We collected CSF specimens from neurosurgical patients with EVD/LD for more than 24 h to perform conventional microbiological studies and mNGS analyses in a pairwise manner. We also investigated the usefulness of mNGS of CSF for the diagnosis of EVD/LD-associated VM. In total, 102 patients were enrolled in this study and divided into three groups, including confirmed VM (cVM) (39), suspected VM (sVM) (49), and non-VM (nVM) (14) groups. Of all the patients, mNGS detected 21 Gram-positive bacteria, 20 Gram-negative bacteria, and five fungi. The three primary bacteria detected were Staphylococcus epidermidis (9), Acinetobacter baumannii (5), and Staphylococcus aureus (3). The mNGS-positive coincidence rate of confirmed EVD/LD-associated VM was 61.54% (24/39), and the negative coincidence rate of the nVM group was 100% (14/14). Of 15 VM pathogens not identified by mNGS in the cVM group, eight were negative with mNGS and seven were inconsistent with the conventional microbiological identification results. In addition, mNGS identified pathogens in 22 cases that were negative using conventional methods; of them, 10 patients received a favorable clinical treatment; thus, showing the benefit of mNGS-guided therapy.

scholarly journals A formalin-free method for stabilizing cells for nucleic acid amplification, hybridization and next-generation sequencing

2015 ◽  
Vol 8 (1) ◽  
Author(s):  
Jianbing Qin ◽  
Jennifer N. Sanmann ◽  
Jeff S. Kittrell ◽  
Pamela A. Althof ◽  
Erin E. Kaspar ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Next Generation ◽  
Generation Sequencing
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