A formalin-free method for stabilizing cells for nucleic acid amplification, hybridization and next-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.

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

10.21203/rs.2.9537/v1 ◽

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.

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Metagenomic Next-Generation Sequencing of Bloodstream Microbial Cell-Free Nucleic Acid in Children With Suspected Sepsis in Pediatric Intensive Care Unit

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.665226 ◽

2021 ◽

Vol 11 ◽

Author(s):

Gangfeng Yan ◽

Jing Liu ◽

Weiming Chen ◽

Yang Chen ◽

Ye Cheng ◽

...

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

Next Generation Sequencing ◽

Nucleic Acid ◽

Pediatric Intensive Care Unit ◽

Pediatric Intensive Care ◽

Microbial Cell ◽

Next Generation ◽

Suspected Sepsis ◽

Generation Sequencing

Bloodstream infection is a life-threatening complication in critically ill patients. Multi-drug resistant bacteria or fungi may increase the risk of invasive infections in hospitalized children and are difficult to treat in intensive care units. The purpose of this study was to use metagenomic next-generation sequencing (mNGS) to understand the bloodstream microbiomes of children with suspected sepsis in a pediatric intensive care unit (PICU). mNGS were performed on microbial cell-free nucleic acid from 34 children admitted to PICU, and potentially pathogenic microbes were identified. The associations of serological inflammation indicators, lymphocyte subpopulations, and other clinical phenotypes were also examined. mNGS of blood samples from children in PICU revealed potential eukaryotic microbial pathogens. The abundance of Pneumocystis jirovecii was positively correlated with a decrease in total white blood cell count and immunodeficiency. Hospital-acquired pneumonia patients showed a significant increase in blood bacterial species richness compared with community-acquired pneumonia children. The abundance of bloodstream bacteria was positively correlated with serum procalcitonin level. Microbial genome sequences from potential pathogens were detected in the bloodstream of children with suspected sepsis in PICU, suggesting the presence of bloodstream infections in these children.

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Finding Pathogenic Nucleic Acid Sequences in Next Generation Sequencing Data

Current Protocols in Human Genetics ◽

10.1002/0471142905.hg1809s86 ◽

2015 ◽

Vol 86 (1) ◽

Author(s):

Michael Parfenov ◽

J.G. Seidman

Keyword(s):

Next Generation Sequencing ◽

Nucleic Acid ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Generation Sequencing ◽

Nucleic Acid Sequences

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PO-086 An efficient ion torrent™ next generation sequencing workflow for liquid biopsy research to assess cell-free total nucleic acid

10.1136/esmoopen-2018-eacr25.614 ◽

2018 ◽

Author(s):

Y Li ◽

K Lea ◽

P Kshatriya ◽

R Cao ◽

J Gu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Nucleic Acid ◽

Liquid Biopsy ◽

Ion Torrent ◽

Total Nucleic Acid ◽

Next Generation ◽

Generation Sequencing

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Mark Roskey on "Sample Prep, Workflow Automation and Advanced Nucleic Acid Fractionation for Next Generation Sequencing"

SciVee ◽

10.4016/19235.01 ◽

2010 ◽

Keyword(s):

Next Generation Sequencing ◽

Nucleic Acid ◽

Next Generation ◽

Workflow Automation ◽

Generation Sequencing

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Study of Preanalytic and Analytic Variables for Clinical Next-Generation Sequencing of Circulating Cell-Free Nucleic Acid

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2017.03.003 ◽

2017 ◽

Vol 19 (4) ◽

pp. 514-524 ◽

Cited By ~ 17

Author(s):

Meenakshi Mehrotra ◽

Rajesh R. Singh ◽

Wei Chen ◽

Richard S.P. Huang ◽

Alaa A. Almohammedsalim ◽

...

Keyword(s):

Next Generation Sequencing ◽

Nucleic Acid ◽

Next Generation ◽

Generation Sequencing

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Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR

BMC Medical Genomics ◽

10.1186/s12920-019-0610-8 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 2

Author(s):

Yuki Nagakubo ◽

Yosuke Hirotsu ◽

Kenji Amemiya ◽

Toshio Oyama ◽

Hitoshi Mochizuki ◽

...

Keyword(s):

Next Generation Sequencing ◽

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Sanger Sequencing ◽

Colorectal Cancers ◽

Next Generation ◽

Braf Mutations ◽

Generation Sequencing

Abstract Background Patients with metastatic colorectal cancer can benefit from anti-EGFR therapy, such as cetuximab and panitumumab. However, colorectal cancers harboring constitutive activating mutations in KRAS, NRAS and BRAF genes are not responsive to anti-EGFR therapy. To select patients for appropriate treatment, genetic testing of these three genes is routinely performed. Methods We applied bridged nucleic acid-clamp real-time PCR (BNA-clamp PCR) to detect somatic hotspot mutations in KRAS, NRAS and BRAF. PCR products from BNA-clamp PCR were subsequently analyzed Sanger sequencing. We then compared results with those from the PCR–reverse sequence-specific oligonucleotide probe (PCR-rSSO) method, which has been used as in vitro diagnostic test in Japan. To validate the mutation status, we also performed next generation sequencing using all samples. Results In 50 formalin-fixed paraffin-embedded tissues, KRAS mutations were detected at frequencies of 50% (25/50) and 52% (26/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively, and NRAS mutations were detected at 12% (6/50) and 12% (6/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively. The concordance rate for detection of KRAS and NRAS mutations between the two was 94% (47/50). However, there were three discordant results. We validated these three discordant and 47 concordant results by next generation sequencing. All mutations identified by BNA-clamp PCR with Sanger sequencing were also identified by next generation sequencing. BNA-clamp PCR detected BRAF mutations in 6% (3/50) of tumor samples. Conclusions Our results indicate that BNA-clamp PCR with Sanger sequencing detects somatic mutations in KRAS, NRAS and BRAF with high accuracy.

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Mitochondrial mutations in patients with congenital heart defects by next generation sequencing technology

Cardiology in the Young ◽

10.1017/s1047951114000754 ◽

2014 ◽

Vol 25 (4) ◽

pp. 705-711 ◽

Cited By ~ 7

Author(s):

Neslihan Abaci ◽

Muzaffer Arıkan ◽

Türkan Tansel ◽

Nazlı Sahin ◽

Aris Cakiris ◽

...

Keyword(s):

Congenital Heart Disease ◽

Heart Disease ◽

Next Generation Sequencing ◽

Nucleic Acid ◽

Heart Development ◽

Congenital Heart ◽

Heart Defects ◽

Next Generation ◽

Oxidative Energy Metabolism ◽

Generation Sequencing

AbstractIt has been shown that mitochondrial deoxyribo nucleic acid mutations may play an important role in the development of cardiomyopathy, and various types of cardiomyopathy can be attributed to disturbed mitochondrial oxidative energy metabolism. Several studies have described many mutations in mitochondrial genes encoding for subunits of respiratory chain complexes. Thus, recent studies confirm that pathologic mitochondrial deoxyribo nucleic acid mutations are a major reason of diseases and determining them by next-generation sequencing will improve our understanding of dysregulation of heart development. To analyse mitochondrial deoxyribo nucleic acid mutations, the entire mitochondrial deoxyribo nucleic acid was amplified in two overlapping polymerase chain reaction fragments from the cardiac tissue of the 22 patients with congenital heart disease, undergoing cardiac surgery. Mitochondrial deoxyribo nucleic acid was deep sequenced by next-generation sequencing. A total of 13 novel mitochondrial deoxyribo nucleic acid mutations were identified in nine patients. Of the patients, three have novel mutations together with reported cardiomyopathy mutations. In all, 65 mutations were found, and 13 of them were unreported. This study represents the most comprehensive mitochondrial deoxyribo nucleic acid mutational analysis in patients with congenital heart disease.

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