Next-generation Sequencing und hochparallele Genexpressionsanalyse in der klinischen Diagnostik / Next-generation sequencing and massively parallel analysis of gene expression: uses in clinical diagnostics

Abstract Background: For the past 30 years, the Sanger method has been the dominant approach and gold standard for DNA sequencing. The commercial launch of the first massively parallel pyrosequencing platform in 2005 ushered in the new era of high-throughput genomic analysis now referred to as next-generation sequencing (NGS). Content: This review describes fundamental principles of commercially available NGS platforms. Although the platforms differ in their engineering configurations and sequencing chemistries, they share a technical paradigm in that sequencing of spatially separated, clonally amplified DNA templates or single DNA molecules is performed in a flow cell in a massively parallel manner. Through iterative cycles of polymerase-mediated nucleotide extensions or, in one approach, through successive oligonucleotide ligations, sequence outputs in the range of hundreds of megabases to gigabases are now obtained routinely. Highlighted in this review are the impact of NGS on basic research, bioinformatics considerations, and translation of this technology into clinical diagnostics. Also presented is a view into future technologies, including real-time single-molecule DNA sequencing and nanopore-based sequencing. Summary: In the relatively short time frame since 2005, NGS has fundamentally altered genomics research and allowed investigators to conduct experiments that were previously not technically feasible or affordable. The various technologies that constitute this new paradigm continue to evolve, and further improvements in technology robustness and process streamlining will pave the path for translation into clinical diagnostics.

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Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows

PLoS ONE ◽

10.1371/journal.pone.0022953 ◽

2011 ◽

Vol 6 (7) ◽

pp. e22953 ◽

Cited By ~ 31

Author(s):

Stefan Siebert ◽

Mark D. Robinson ◽

Sophia C. Tintori ◽

Freya Goetz ◽

Rebecca R. Helm ◽

...

Keyword(s):

Gene Expression ◽

Next Generation Sequencing ◽

Differential Gene Expression ◽

Next Generation ◽

Differential Gene ◽

Generation Sequencing

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Erratum to: Evaluating next-generation sequencing for direct clinical diagnostics in diarrhoeal disease

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-017-2993-9 ◽

2017 ◽

Vol 36 (7) ◽

pp. 1339-1342

Author(s):

K. G. Joensen ◽

A. L. Ø. Engsbro ◽

O. Lukjancenko ◽

R. S. Kaas ◽

O. Lund ◽

...

Keyword(s):

Next Generation Sequencing ◽

Diarrhoeal Disease ◽

Clinical Diagnostics ◽

Next Generation ◽

Generation Sequencing

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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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DNA Methylation Landscape of 16 Canine Somatic Tissues by Methylation-Sensitive Restriction Enzyme-Based Next Generation Sequencing

10.21203/rs.3.rs-146319/v1 ◽

2021 ◽

Author(s):

Jumpei Yamazaki ◽

Yuki Matsumoto ◽

Jaroslav Jelinek ◽

Teita Ishizaki ◽

Shingo Maeda ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Next Generation Sequencing ◽

Restriction Enzyme ◽

Companion Animals ◽

Next Generation ◽

Cpg Sites ◽

Somatic Tissues ◽

Methylation Sensitive Restriction Enzyme ◽

Generation Sequencing

Abstract Background: DNA methylation plays important functions in gene expression regulation that is involved in individual development and various diseases. DNA methylation has been well studied in human and model organisms, but only limited data exist in companion animals like dog. Results: Using methylation-sensitive restriction enzyme-based next generation sequencing (Canine DREAM), we obtained canine DNA methylation maps from 16 somatic tissues. In total, we evaluated 130,861 CpG sites. The majority of CpG sites were either highly methylated (>70%, 52.5%-64.6% of all CpG sites analyzed) or unmethylated (<30%, 22.5%-28.0% of all CpG sites analyzed) which are methylation patterns similar to other species. The overall methylation status of CpG sites across the 32 methylomes were remarkably similar. However, the tissue types were clearly defined by principle component analysis and hierarchical clustering analysis with DNA methylome. We found 6416 CpG sites located closely at promoter region of genes and inverse correlation between DNA methylation and gene expression of these genes. Conclusions: Our study provides basic dataset for DNA methylation profiles in dogs.

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