scholarly journals Profiling of the Causative Bacteria in Infected Lymphocysts after Lymphadenectomy for Gynecologic Cancer by Pyrosequencing the 16S Ribosomal RNA Gene Using Next-Generation Sequencing Technology

2019 ◽  
Vol 2019 ◽  
pp. 1-5 ◽  
Author(s):  
Yuya Nogami ◽  
Kouji Banno ◽  
Masataka Adachi ◽  
Haruko Kunitomi ◽  
Yusuke Kobayashi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pathogenic Bacteria ◽  
Sequence Data ◽  
Bacterial Flora ◽  
Gynecologic Cancer ◽  
False Negative ◽  
Enteric Bacteria ◽  
Next Generation ◽  
Single Strain ◽  
Generation Sequencing

Background. Surgery for gynecologic cancer with lymphadenectomy and pelvic radiotherapy can produce lymphoceles that sometimes complicate with infection, resulting in abscesses. The true pathogenic bacteria of abscesses are not always found because of false-negative results due to administered antibiotics and difficulty with detection, including for anaerobic bacteria. Analyzing bacteria flora by next-generation sequencing (NGS) using 16S ribosomal DNA may reveal the true pathogenic bacteria in abscesses. This is the first report on causative pathogens for infectious lymphocele using this technology. Methods. The subjects were patients who developed infectious lymphocele after surgery for gynecologic cancer at our hospital from July 2015 to September 2016. NGS analyses of bacterial flora were performed using specimens preserved at -80°C. Two steps of PCR were performed for purified DNA samples to obtain sequence libraries. Processing of sequence data, including operational taxonomic unit (OTU) definition, taxonomy assignment, and an OTU BLAST search were performed. All patients gave written informed consent and the study was approved by the institutional research ethics committee. Results. Six patients underwent puncture and drainage. The result in most cases indicated a single causative pathogen, including Staphylococcus lugdunensis, Streptococcus dysgalactiae, Streptococcus equinus, Enterococcus saccharolyticus, and Escherichia coli. Conclusions. NGS revealed that the causative bacteria in lymphocele infection are normally a single strain, such as a surface Gram-positive coccus or enteric bacteria. Antibiotics should be chosen as appropriate for elimination of these respective bacteria.

scholarly journals Clinical Analysis of Metagenomic Next-generation Sequencing Confirmed Chlamydia psittaci Pneumonia: A Case Series and Literature Review

2021 ◽  
Author(s):  
Xin-Qi Teng ◽  
Wen-Cheng Gong ◽  
Ting-Ting Qi ◽  
Guo-Hua Li ◽  
Qiang Qu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Literature Review ◽  
Clinical Characteristics ◽  
Pathogenic Bacteria ◽  
False Negative ◽  
False Negative Rate ◽  
Case Series ◽  
Chlamydia Psittaci ◽  
Next Generation ◽  
Generation Sequencing

Abstract Introduction: Chlamydia psittaci infection is a zoonotic infectious disease, which mainly inhaled through the lungs when exposed to the secretions of poultry that carry pathogenic bacteria. The traditional respiratory specimens or serological antibody testing is slow and the false-negative rate is high. Metagenomic next-generation sequencing gives a promising rapid diagnosis tool. Methods: We retrospective summarized the clinical characteristics of five C. psittaci pneumonia patients diagnosed by mNGS, conducted a literature review summarizing the clinical characteristics of patients with C. psittaci pneumonia reported since 2010.Results: Five C. psittaci. pneumonia patients confirmed by mNGS aged from 36 to 66 years with three males. 60% of patients had type 2 diabetes mellitus. And 60% of patients had a history of contact with avian or poultry. All patients had a high fever over 38.5 °C, cough, hypodynamia, hypoxemia, and dyspnea on admission. Two patients had invasive ventilator support and Extracorporeal Membrane Oxygenation support. The levels of C-reactive protein, procalcitonin, and erythrocyte sedimentation rate on admission and follow-up were all higher than normal values. Doxycycline or moxifloxacin monotherapy was accounted for 1/5 (20%) and 2/5 (40%) patients, and combination therapy was accounted for 2/5(40%) patients. Four patients improved and were discharged, and one patient died due to multiple organ failure and disseminated intravascular coagulation.Conclusions: mNGS can increase the detection rate of C. psittaci, shorten the diagnosis time of C. psittaci pneumonia and improve the prognosis of patients.

scholarly journals Next-generation sequencing analysis of bacterial flora in bovine protothecal mastitic milk and feces

2019 ◽  
Vol 81 (11) ◽  
pp. 1547-1551 ◽  
Author(s):  
Ayumi MIURA ◽  
Tomomi KURUMISAWA ◽  
Rui KANO ◽  
Takaaki ITO ◽  
Kazuyuki SUZUKI ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Bacterial Flora ◽  
Sequencing Analysis ◽  
Next Generation ◽  
Next Generation Sequencing Analysis ◽  
Mastitic Milk ◽  
Generation Sequencing

scholarly journals Validation of variants using cost effective highresolution melting (HRM) analysis predicted from target re-sequencing in Eucalyptus

2020 ◽  
Vol 79 (2) ◽  
pp. 105-113
Author(s):  
Abdul Bari Muneera Parveen ◽  
Divya Lakshmanan ◽  
Modhumita Ghosh Dasgupta
Keyword(s):  
Next Generation Sequencing ◽  
Large Scale ◽  
Sequence Data ◽  
Cost Effective ◽  
Nucleotide Polymorphisms ◽  
Next Generation ◽  
Time Saving ◽  
Hrm Analysis ◽  
The Cost ◽  
Generation Sequencing

The advent of next-generation sequencing has facilitated large-scale discovery and mapping of genomic variants for high-throughput genotyping. Several research groups working in tree species are presently employing next generation sequencing (NGS) platforms for marker discovery, since it is a cost effective and time saving strategy. However, most trees lack a chromosome level genome map and validation of variants for downstream application becomes obligatory. The cost associated with identifying potential variants from the enormous amount of sequence data is a major limitation. In the present study, high resolution melting (HRM) analysis was optimized for rapid validation of single nucleotide polymorphisms (SNPs), insertions or deletions (InDels) and simple sequence repeats (SSRs) predicted from exome sequencing of parents and hybrids of Eucalyptus tereticornis Sm. ? Eucalyptus grandis Hill ex Maiden generated from controlled hybridization. The cost per data point was less than 0.5 USD, providing great flexibility in terms of cost and sensitivity, when compared to other validation methods. The sensitivity of this technology in variant detection can be extended to other applications including Bar-HRM for species authentication and TILLING for detection of mutants.

scholarly journals Metagenomic analysis using next-generation sequencing of pathogens in bronchoalveolar lavage fluid from pediatric patients with respiratory failure

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Suguru Takeuchi ◽  
Jun-ichi Kawada ◽  
Kazuhiro Horiba ◽  
Yusuke Okuno ◽  
Toshihiko Okumura ◽  
...  
Keyword(s):  
Respiratory Failure ◽  
Next Generation Sequencing ◽  
Bronchoalveolar Lavage ◽  
Bronchoalveolar Lavage Fluid ◽  
Pediatric Patients ◽  
Respiratory Infections ◽  
Sequence Data ◽  
Lavage Fluid ◽  
Next Generation ◽  
Generation Sequencing

Abstract Next-generation sequencing (NGS) has been applied in the field of infectious diseases. Bronchoalveolar lavage fluid (BALF) is considered a sterile type of specimen that is suitable for detecting pathogens of respiratory infections. The aim of this study was to comprehensively identify causative pathogens using NGS in BALF samples from immunocompetent pediatric patients with respiratory failure. Ten patients hospitalized with respiratory failure were included. BALF samples obtained in the acute phase were used to prepare DNA- and RNA-sequencing libraries. The libraries were sequenced on MiSeq, and the sequence data were analyzed using metagenome analysis tools. A mean of 2,041,216 total reads were sequenced for each library. Significant bacterial or viral sequencing reads were detected in eight of the 10 patients. Furthermore, candidate pathogens were detected in three patients in whom etiologic agents were not identified by conventional methods. The complete genome of enterovirus D68 was identified in two patients, and phylogenetic analysis suggested that both strains belong to subclade B3, which is an epidemic strain that has spread worldwide in recent years. Our results suggest that NGS can be applied for comprehensive molecular diagnostics as well as surveillance of pathogens in BALF from patients with respiratory infection.

scholarly journals Comparison of Normalization Methods for Construction of Large, Multiplex Amplicon Pools for Next-Generation Sequencing

2010 ◽  
Vol 76 (12) ◽  
pp. 3863-3868 ◽  
Author(s):  
J. Kirk Harris ◽  
Jason W. Sahl ◽  
Todd A. Castoe ◽  
Brandie D. Wagner ◽  
David D. Pollock ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Massively Parallel Sequencing ◽  
Sequence Data ◽  
Cost Savings ◽  
Massively Parallel ◽  
Next Generation ◽  
Normalization Methods ◽  
The Cost ◽  
Generation Sequencing

ABSTRACT Constructing mixtures of tagged or bar-coded DNAs for sequencing is an important requirement for the efficient use of next-generation sequencers in applications where limited sequence data are required per sample. There are many applications in which next-generation sequencing can be used effectively to sequence large mixed samples; an example is the characterization of microbial communities where ≤1,000 sequences per samples are adequate to address research questions. Thus, it is possible to examine hundreds to thousands of samples per run on massively parallel next-generation sequencers. However, the cost savings for efficient utilization of sequence capacity is realized only if the production and management costs associated with construction of multiplex pools are also scalable. One critical step in multiplex pool construction is the normalization process, whereby equimolar amounts of each amplicon are mixed. Here we compare three approaches (spectroscopy, size-restricted spectroscopy, and quantitative binding) for normalization of large, multiplex amplicon pools for performance and efficiency. We found that the quantitative binding approach was superior and represents an efficient scalable process for construction of very large, multiplex pools with hundreds and perhaps thousands of individual amplicons included. We demonstrate the increased sequence diversity identified with higher throughput. Massively parallel sequencing can dramatically accelerate microbial ecology studies by allowing appropriate replication of sequence acquisition to account for temporal and spatial variations. Further, population studies to examine genetic variation, which require even lower levels of sequencing, should be possible where thousands of individual bar-coded amplicons are examined in parallel.

Next-generation sequencing in metastatic lung cancer patients: Translation from sequence data into clinical practice.

2015 ◽  
Vol 33 (15_suppl) ◽  
pp. e12521-e12521
Author(s):  
Jessica Ribeiro Gomes ◽  
Raphael Brandao Moreira ◽  
Renata D'Alpino D'Alpino ◽  
Marcelo Rocha S Cruz ◽  
Tercia Tarciane Soares de Sousa ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Clinical Practice ◽  
Next Generation Sequencing ◽  
Cancer Patients ◽  
Sequence Data ◽  
Metastatic Lung Cancer ◽  
Next Generation ◽  
Lung Cancer Patients ◽  
Metastatic Lung ◽  
Generation Sequencing

scholarly journals Use of Next Generation Sequencing to study two cowpox virus outbreaks

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6561 ◽  
Author(s):  
Markus H. Antwerpen ◽  
Enrico Georgi ◽  
Alexandra Nikolic ◽  
Gudrun Zoeller ◽  
Peter Wohlsein ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Group Method ◽  
Common Source ◽  
Cowpox Virus ◽  
Next Generation ◽  
Single Strain ◽  
Hemagglutinin Gene ◽  
Hospital Acquired ◽  
Virus Isolates ◽  
Generation Sequencing

BackgroundBetween 2008 and 2011 about 40 cases of human cowpox were reported from Germany and France. Infections had been acquired via close contact to infected, young pet rats. An identical and unique sequence of the hemagglutinin gene was found in various cowpox virus (CPXV) isolates pointing to a common source of infection. In a second CPXV outbreak in cats in a small animal clinic in Germany in 2015, four out of five hospitalized cats showed identical hemagglutinin sequences and thus, a hospital-acquired transmission had been assumed. Next-Generation Sequencing was performed in order to re-investigate the outbreaks, as epidemiological data could not confirm all cases.MethodsHomogenates of lesion material from rats, cats and humans were cultivated in cell culture. The genomes of four virus isolates, nine CPXVs from our strain collections and from DNA of three paraffin-embedded lesion materials were determined by Next Generation Sequencing (NGS). For phylogenetic analyses a MAFFT-alignment was generated. A distance matrix based on concatenated SNPs was calculated and plotted as dendrogram using Unweighted Pair Group Method with Arithmetic mean (UPGMA) for visualization.ResultsAligning of about 200.000 nucleotides of 8 virus isolates associated with the pet rat outbreak revealed complete identity of six genomes, the remainder two genomes differed in as little as 3 SNPs. When comparing this dataset with four already published CPXV genomes also associated with the pet rat outbreak, again a maximum difference of 3 SNPs was found. The outbreak which lasted from 2008 till 2011 was indeed caused by a single strain which has maintained an extremely high level of clonality over 4 years. Aligning genomic sequences from four cases of feline cowpox revealed 3 identical sequences and one sequence which differed in 65 nucleotides. Although identical hemagglutinin sequences had been obtained from four hospitalized cats, genomic sequencing proved that a hospital-acquired transmission had occurred in only three cats.ConclusionAnalyzing the rather short sequence of the hemagglutinin gene is not sufficient to conduct molecular trace back analyses. Instead, whole genome sequencing is the method of choice which can even be applied to paraffin-embedded specimens.

scholarly journals Hidden biases in germline structural variant detection

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Michael M. Khayat ◽  
Sayed Mohammad Ebrahim Sahraeian ◽  
Samantha Zarate ◽  
Andrew Carroll ◽  
Huixiao Hong ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
False Negative ◽  
False Negative Rate ◽  
Next Generation Sequencing Data ◽  
Chinese Family ◽  
Next Generation ◽  
Sequencing Data ◽  
Structural Variations ◽  
The Impact ◽  
Generation Sequencing

Abstract Background Genomic structural variations (SV) are important determinants of genotypic and phenotypic changes in many organisms. However, the detection of SV from next-generation sequencing data remains challenging. Results In this study, DNA from a Chinese family quartet is sequenced at three different sequencing centers in triplicate. A total of 288 derivative data sets are generated utilizing different analysis pipelines and compared to identify sources of analytical variability. Mapping methods provide the major contribution to variability, followed by sequencing centers and replicates. Interestingly, SV supported by only one center or replicate often represent true positives with 47.02% and 45.44% overlapping the long-read SV call set, respectively. This is consistent with an overall higher false negative rate for SV calling in centers and replicates compared to mappers (15.72%). Finally, we observe that the SV calling variability also persists in a genotyping approach, indicating the impact of the underlying sequencing and preparation approaches. Conclusions This study provides the first detailed insights into the sources of variability in SV identification from next-generation sequencing and highlights remaining challenges in SV calling for large cohorts. We further give recommendations on how to reduce SV calling variability and the choice of alignment methodology.

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