Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota
| Autor: | Maria A. J. de Ridder, Willem de Koning, Chantal B. van Houten, Deborah Horst-Kreft, Rick Jansen, Andrew P. Stubbs, John P. Hays, Astrid P. Heikema, Robert Kraaij, Stefan A. Boers, Saskia Hiltemann, Louis Bont |
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| Přispěvatelé: | Medical Microbiology & Infectious Diseases, Pathology, Internal Medicine, Medical Informatics |
| Rok vydání: | 2020 |
| Předmět: |
Male
0301 basic medicine Corynebacterium Nanopores RNA Ribosomal 16S bacterial species Child Genetics (clinical) 0303 health sciences biology Microbiota Illumina sequencing High-Throughput Nucleotide Sequencing Middle Aged Nanopore Child Preschool 16s rrna gene sequencing Female Base calling Nasal Cavity Adult DNA Bacterial lcsh:QH426-470 Adolescent 030106 microbiology nasal microbiota mothur Computational biology Article Young Adult 03 medical and health sciences Species level Genetics Humans Illumina dye sequencing 030304 developmental biology Aged DNA Primers 030306 microbiology microbiology Computational Biology Infant Genes rRNA biology.organism_classification lcsh:Genetics Nanopore Sequencing 030104 developmental biology 16S rRNA gene Nanopore sequencing |
| Zdroj: | Genes Genes, Vol 11, Iss 1105, p 1105 (2020) Genes, 11(9):E1105. Multidisciplinary Digital Publishing Institute (MDPI) Volume 11 Issue 9 |
| ISSN: | 2073-4425 |
| DOI: | 10.3390/genes11091105 |
| Popis: | 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 swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME (Oxford Nanopore Technologies&mdash ONT, Oxford, UK) and an in-house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 (Oxford Nanopore Technologies&mdash ONT, Oxford, UK) and R9.2 (Oxford Nanopore Technologies&mdash ONT, Oxford, UK) were compared. In conclusion, the current study shows that the nanopore sequencing platform is comparable with the Illumina platform in detection bacterial genera of the nasal microbiota, but the nanopore platform does have problems in detecting bacteria within the genus Corynebacterium. Although advances are being made, thorough validation of the nanopore platform is still recommendable. |
| Databáze: | OpenAIRE |
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