Sensitivity of Next-Generation Sequencing Metagenomic Analysis for Detection of RNA and DNA Viruses in Cerebrospinal Fluid: The Confounding Effect of Background Contamination

Bukowska-Ośko, Iwona; Perlejewski, Karol; Nakamura, Shota; Motooka, Daisuke; Stokowy, Tomasz; Kosińska, Joanna; Popiel, Marta; Płoski, Rafał; Horban, Andrzéj; Lipowski, Dariusz; Cortés, Kamila Caraballo; Pawełczyk, Agnieszka; Demkow, Urszula; Stępień, Adam; Radkowski, Marek; Laskus, Tomasz

doi:10.1007/5584_2016_138

Cited by 9 publications

(8 citation statements)

References 41 publications

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“…Further comparative efforts, including ones aimed at a determination of limits of detection (Bukowska-Ośko et al, 2017) are clearly needed to clarify the picture on the analytical sensitivity of HTS approaches.…”

Section: Considerations For An Hts Detection Assaymentioning

confidence: 99%

“…In the case of biological indexing, sometimes considered the gold standard in woody host plants, two large studies in grapevine (Al Rwahnih et al, 2015 ) and in temperate fruit trees (Rott et al, 2017 ) also suggest comparable sensitivities. Further comparative efforts, including ones aimed at a determination of limits of detection (Bukowska-Ośko et al, 2017 ) are clearly needed to clarify the picture on the analytical sensitivity of HTS approaches.…”

Section: Considerations For An Hts Detection Assaymentioning

confidence: 99%

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Application of HTS for Routine Plant Virus Diagnostics: State of the Art and Challenges

et al. 2018

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Section: Considerations For An Hts Detection Assaymentioning

confidence: 99%

Section: Considerations For An Hts Detection Assaymentioning

confidence: 99%

Application of HTS for Routine Plant Virus Diagnostics: State of the Art and Challenges

et al. 2018

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“…During previous studies, we encountered the problem of contaminating DNA complicating the analysis of sequencing results (26), which is emerging as a common problem encountered when attempting to do metagenomic analysis of clinical samples (3,(27)(28)(29). Here, we evaluated three commercially available WGA kits to characterize the relative amount and composition of contaminating DNA associated with the use of each of them to inform selection of the appropriate WGA kit for further metagenomics studies of PJI.…”

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confidence: 99%

Impact of Contaminating DNA in Whole-Genome Amplification Kits Used for Metagenomic Shotgun Sequencing for Infection Diagnosis

et al. 2017

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Whole-genome amplification (WGA) is a useful tool for amplification of very small quantities of DNA for many uses, including metagenomic shotgun sequencing for infection diagnosis. Depending on the application, background DNA from WGA kits can be problematic. Three WGA kits were tested for their utility in a metagenomics approach to identify the pathogens in sonicate fluid comprised of biofilms and other materials dislodged from the surfaces of explanted prosthetic joints using sonication. The Illustra V2 Genomiphi, Illustra single cell Genomiphi, and Qiagen REPLI-g single cell kits were used to test identical sonicate fluid samples. Variations in the number of background reads, the genera identified in the background, and the number of reads from known pathogens known to be present in the samples were observed between kits. These results were then compared to those obtained with a library preparation without prior WGA using an NEBNext Ultra II paired-end kit, which requires a very small amount of input DNA. This approach also resulted in the presence of contaminant bacterial DNA and yielded fewer reads from the known pathogens. These findings highlight the impact that WGA kit selection can have on metagenomic analysis of low-biomass samples and the importance of the careful selection and consideration of the implications of using these tools.KEYWORDS metagenomics, prosthetic joint infection, whole-genome amplification M etagenomic shotgun sequencing is a rapidly developing powerful tool to examine microbial communities. It is also gaining interest in the field of clinical microbiology as a way to identify pathogens in normally sterile specimens (1-7). Not only does this approach allow for pathogen identification, but the gene content information can also be used for other analyses, such as antibiotic resistance prediction (8, 9), typing for tracking of outbreaks or epidemiological studies (10), or assessment for other relevant genes, such as those encoding virulence factors (11,12).One significant barrier to metagenomic shotgun sequencing approaches is the amount of genetic material necessary to perform these experiments. This is not an obstacle in settings such as fecal microbiome studies, where abundant organisms are present, but in other scenarios (e.g., in scenarios with low-biomass environmental samples and uncultured bacteria or in single cell analysis), the number of cells and the amount of genetic material can be limited (13)(14)(15)(16)(17). It is in these situations that whole-genome amplification (WGA) has the potential to play an important role in pathogen detection. Citation Thoendel M, Jeraldo P, GreenwoodQuaintance KE, Yao J, Chia N, Hanssen AD, Abdel MP, Patel R. 2017. Impact of contaminating DNA in whole-genome amplification kits used for metagenomic shotgun sequencing for infection diagnosis. J

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“…This inconsistency could be due to general lower sensitivity of metagenomic workflows compared with real-time RT-PCR/PCR assays. Using serial dilutions of HIV- and HSV-positive sera in negative CSF, we previously established that the limit of detection for RNA- and DNA-based metagenomics was no better than 10 2 and 10 3 viral copies/reaction, respectively [ 60 ].…”

Section: Discussionmentioning

confidence: 99%

Search for Viral Infections in Cerebrospinal Fluid From Patients With Autoimmune Encephalitis

Perlejewski

Bukowska-Ośko

Rydzanicz

et al. 2020

Open Forum Infectious Diseases

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Background It has been reported that a virus-mediated brain tissue damage can lead to autoimmune encephalitis (AE) characterized by the presence of antibodies against neuronal surface antigens. In the study we investigate the presence of viruses in cerebrospinal fluid (CSF) from patients with AE using RT-PCR/PCR and shotgun metagenomics. Methods CSF samples collected from two hundred patients with encephalitis were tested for the presence of antibodies against anti-glutamate receptor (NMDAR), contactin-associated protein 2 (CASPR2), glutamate receptors (type AMPA1/2), leucine-rich glioma-inactivated protein 1 (LGI1), dipeptidyl aminopeptidase-like protein 6 (DPPX) and GABA B receptor and those found positive were further analyzed with real-time RT-PCR/PCR for common viral neuroinfections and shotgun DNA- and RNA-based metagenomics. Results Autoantibodies against neuronal cells were detected in CSF from 8 individuals (4% of all encephalitis patients): 7 (3.5%) had anti-NMDAR and one (0.5%) had anti-GABA B. RT-PCR/PCR identified human herpes virus type 1 (HSV-1); (300 copies/ml) and the representative of Enterovirus genus (550 copies/ml) in one patient each. Torque teno virus (TTV) was found in another patient using metagenomic analysis and its presence was confirmed by specific PCR. Conclusions We detected the presence of HSV, TTV and Enterovirus genus in CSF samples from 3 out of 8 AE patients. These findings support the concept of viral involvement in the pathogenesis of this disease.

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Sensitivity of Next-Generation Sequencing Metagenomic Analysis for Detection of RNA and DNA Viruses in Cerebrospinal Fluid: The Confounding Effect of Background Contamination

Cited by 9 publications

References 41 publications

Application of HTS for Routine Plant Virus Diagnostics: State of the Art and Challenges

Application of HTS for Routine Plant Virus Diagnostics: State of the Art and Challenges

Impact of Contaminating DNA in Whole-Genome Amplification Kits Used for Metagenomic Shotgun Sequencing for Infection Diagnosis

Search for Viral Infections in Cerebrospinal Fluid From Patients With Autoimmune Encephalitis

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