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Illumina 与 Nanopore 16S rRNA 基因测序技术在人类鼻腔微生物组中的比较。

Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota.

机构信息

Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center (Erasmus MC), 3015 CN Rotterdam, The Netherlands.

Department of Microbiology, Leiden University Medical Center (LUMC), 2333 ZA Leiden, The Netherlands.

出版信息

Genes (Basel). 2020 Sep 21;11(9):1105. doi: 10.3390/genes11091105.

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 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-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 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 and a considerable part to the genus 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-ONT, Oxford, UK) and R9.2 (Oxford Nanopore Technologies-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 . Although advances are being made, thorough validation of the nanopore platform is still recommendable.

摘要

Illumina 和纳米孔测序技术是强大的工具,可用于确定复杂微生物群落中的细菌组成。在这项研究中,我们比较了使用 Illumina 和纳米孔 16S rRNA 基因测序在属水平上的鼻腔微生物组结果。我们还使用纯单种培养物监测纳米孔测序在准确鉴定物种方面的进展,并评估了纳米孔 EPI2ME 16S 数据分析管道的性能。使用 Illumina MiSeq 和牛津纳米孔 16S rRNA 基因测序技术对 59 个鼻腔拭子进行了测序。此外,还使用纳米孔测序技术对五种相关细菌的纯培养物进行了测序。Illumina MiSeq 序列数据使用 Mothur 软件包中提供的生物信息学模块进行处理。Albacore 和 Guppy 碱基调用、纳米孔 EPI2ME(牛津纳米孔技术公司-ONT,英国牛津)中的工作流程以及内部开发的生物信息学脚本用于分析纳米孔数据。在属水平上,发现了相似的细菌多样性谱,并且两种平台都鉴定了五个主要和已建立的属。然而,可能由于纳米孔序列引物不匹配,纳米孔测序平台鉴定的 属的丰度要低得多。此外,当在 EPI2ME 工作流程中使用默认设置时,几乎所有似乎属于细菌属 和相当一部分属 的序列读取仅在科水平上被鉴定。对单种培养物的纳米孔测序表明,对于测试的 5 个菌株中的 4 个,至少可以在属和种水平上准确识别 88%的物种,包括当比较碱基调用器 Guppy 和 Albacore 以及当比较通量版本 R9.4(牛津纳米孔技术公司-ONT,英国牛津)和 R9.2(牛津纳米孔技术公司-ONT,英国牛津)时,准确序列读取识别的改进。总之,本研究表明,纳米孔测序平台在检测鼻腔微生物群的细菌属方面与 Illumina 平台相当,但纳米孔平台在检测属内细菌方面确实存在问题。尽管取得了进展,但仍建议对纳米孔平台进行彻底验证。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ef42/7565314/b0e4c8a9f656/genes-11-01105-g001.jpg

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