Abstract:Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal… Show more
“…Literature shows that the use of Oxford Nanopore's commercial barcoding primers also potentially interfere with one's capacity to detect certain bacterial taxa in complex samples. Corynebacterium, Pseudomonas and Bifidobacterium have been reported to be some of the affected genera (9,10,25).…”
Section: Discussionmentioning
confidence: 99%
“…Moreover, size range cutoffs chosen to filter reads certainly have an impact on the number of reads retrieved in a given experiment. Thus, most reports for microbial community analysis using 16S we found applied different Q Score thresholds (usually Q Score > 7) and variable cutoff values (e.g., 1,300-1,950 bp, <1,700 bp, 1,400-1,700 bp, and 1,350-1,650 bp) (9,10,25,27).…”
Section: Discussionmentioning
confidence: 99%
“…While promising, 16S-based species-level microbial profiling using nanopore remains a rather controversial topic. Current error rates reported for long-read basecalling algorithms are usually higher than the differences between closely related species, which makes species-level assignment unadvisable due to the possibility of false positives (9,10). Using a mock community, Winand and collaborators reported that, even though genuslevel classification of nanopore is highly accurate, the use of absolute number of reads as a "tiebreak" between organisms of the same genus would not reflect the real composition of the community for all present species (9).…”
Section: Discussionmentioning
confidence: 99%
“…Despite gaining traction, nanopore sequencing protocols and analysis pipelines are still being validated by the community at large, and many research groups have been thoroughly evaluating its performance against traditional (usually Illumina) platforms (9)(10)(11)(12). In the present study, we investigated how nanoporebased long-read sequencing compares to Illumina sequencing for the study of complex microbial communities from hospital surfaces.…”
Fast and accurate identification of pathogens is an essential task in healthcare settings. Second-generation sequencing platforms such as Illumina have greatly expanded the capacity with which different organisms can be detected in hospital samples, and third-generation nanopore-driven sequencing devices such as Oxford Nanopore's minION have recently emerged as ideal sequencing platforms for routine healthcare surveillance due to their long-read capacity and high portability. Despite its great potential, protocols and analysis pipelines for nanopore sequencing are still being extensively validated. In this work, we assess the ability of nanopore sequencing to provide reliable community profiles based on 16S rRNA sequencing in comparison to traditional Illumina platforms using samples collected from Intensive Care Units of a hospital in Brazil. While our results demonstrate that lower throughputs may be a shortcoming of the method in more complex samples, we show that the use of single-use Flongle flowcells in nanopore sequencing runs can provide insightful information on the community composition in healthcare settings.
“…Literature shows that the use of Oxford Nanopore's commercial barcoding primers also potentially interfere with one's capacity to detect certain bacterial taxa in complex samples. Corynebacterium, Pseudomonas and Bifidobacterium have been reported to be some of the affected genera (9,10,25).…”
Section: Discussionmentioning
confidence: 99%
“…Moreover, size range cutoffs chosen to filter reads certainly have an impact on the number of reads retrieved in a given experiment. Thus, most reports for microbial community analysis using 16S we found applied different Q Score thresholds (usually Q Score > 7) and variable cutoff values (e.g., 1,300-1,950 bp, <1,700 bp, 1,400-1,700 bp, and 1,350-1,650 bp) (9,10,25,27).…”
Section: Discussionmentioning
confidence: 99%
“…While promising, 16S-based species-level microbial profiling using nanopore remains a rather controversial topic. Current error rates reported for long-read basecalling algorithms are usually higher than the differences between closely related species, which makes species-level assignment unadvisable due to the possibility of false positives (9,10). Using a mock community, Winand and collaborators reported that, even though genuslevel classification of nanopore is highly accurate, the use of absolute number of reads as a "tiebreak" between organisms of the same genus would not reflect the real composition of the community for all present species (9).…”
Section: Discussionmentioning
confidence: 99%
“…Despite gaining traction, nanopore sequencing protocols and analysis pipelines are still being validated by the community at large, and many research groups have been thoroughly evaluating its performance against traditional (usually Illumina) platforms (9)(10)(11)(12). In the present study, we investigated how nanoporebased long-read sequencing compares to Illumina sequencing for the study of complex microbial communities from hospital surfaces.…”
Fast and accurate identification of pathogens is an essential task in healthcare settings. Second-generation sequencing platforms such as Illumina have greatly expanded the capacity with which different organisms can be detected in hospital samples, and third-generation nanopore-driven sequencing devices such as Oxford Nanopore's minION have recently emerged as ideal sequencing platforms for routine healthcare surveillance due to their long-read capacity and high portability. Despite its great potential, protocols and analysis pipelines for nanopore sequencing are still being extensively validated. In this work, we assess the ability of nanopore sequencing to provide reliable community profiles based on 16S rRNA sequencing in comparison to traditional Illumina platforms using samples collected from Intensive Care Units of a hospital in Brazil. While our results demonstrate that lower throughputs may be a shortcoming of the method in more complex samples, we show that the use of single-use Flongle flowcells in nanopore sequencing runs can provide insightful information on the community composition in healthcare settings.
“…And MinION and Illumina platforms generated comparable results from different biofluids including human nasal samples. 25 Many metagenomics studies adopt commercial kits for DNA extraction and reports demonstrated that manual method processed fecal samples better captures the microbial diversity. 26 In this study we compared two manual methods of DNA extraction for further processing.…”
In the present era, emergence of next generation sequencing approaches has revolutionized the field of gut microbiome study. However, the adopted DNA extraction step used in metagenomics experiments and its efficiency may play a critical role in their reproducibility and outcome. In this study, fecal samples from active and non-tuberculosis subjects (ATB/NTB, n=7) were used. Fecal samples of a subgroup of these subjects were subjected to Mechanical enzymatic lysis (MEL) and Phenol: Chloroform: Isoamyl Alcohol (PCIA) methods of DNA extraction and a third-generation sequencing platform i.e. MinION was employed for microbiome profiling. Findings of this study demonstrated that DNA extraction method significantly impacts the DNA yield and microbial diversity. Irrespective of the adopted method of DNA extraction, ATB patients showed altered microbial diversity compared to NTB controls. Also, the fecal microbial diversity details are better captured in samples processed by MEL method and may be suitable to be adopted for high-throughput gut microbiome studies.
Objective: Nosocomial bacterial meningitis is one of the major complications after neurosurgery. We performed nanopore 16S amplicon sequencing from cerebrospinal fluid (CSF) to evaluate bacterial meningitis in patients who underwent neurosurgery. Methods: Among the patients who visited the neurosurgery department of Seoul National University Hospital between July 2017 and June 2020, those with clinically suspected bacterial meningitis were included. 16S rDNA PCR was performed from the CSF, and nanopore sequencing was performed for up to 3 h. The reads were aligned to the BLAST database. In each case, the culture and the 16S rRNA gene amplicon analysis were simultaneously performed and compared with each other, and we retrospectively reviewed the medical records. Genuine infection was determined by the identical results between conventional culture study and the sequencing, or clinically determined in cases with inconsistent results between the two methods. Results: Of the 285 samples obtained from 178 patients who had 16S rDNA PCR, 41 samples (14.4%) were diagnosed with genuine infection. A total of 56.1% (23/41) of the samples with genuine infection showed a false-negative culture test. In particular, 16S amplicon sequencing was useful in evaluating patients at the initial tests who had infection with intraventricular hemorrhage (Culture false-negative rate = 100%), subarachnoid hemorrhage (Culture falsenegative rate = 77.8%), and systemic cancer (Culture false-negative rate = 100%), which are risk factors for central fever. Moreover, 16S amplicon sequencing could suggest the possibility of persistent bacterial meningitis in empirical antibiotic use. Conclusion: CSF nanopore 16S sequencing was more effective than conventional CSF culture studies in postoperative bacterial meningitis and may contribute to evidence-based decisions for antibiotic maintenance and discontinuation.
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