PDF(Pubmed)
Abstract：
16S rRNA targeted amplicon sequencing is an established standard for elucidating microbial community composition. While high-throughput short-read sequencing can elicit only a portion of the 16S rRNA gene due to their limited read length, third generation sequencing can read the 16S rRNA gene in its entirety and thus provide more precise taxonomic classification. Here, we present a protocol for generating full-length 16S rRNA sequences with Oxford Nanopore Technologies (ONT) and a microbial community profile with Emu. We select Emu for analyzing ONT sequences as it leverages information from the entire community to overcome errors due to incomplete reference databases and hardware limitations to ultimately obtain species-level resolution. This pipeline provides a low-cost solution for characterizing microbiome composition by exploiting real-time, long-read ONT sequencing and tailored software for accurate characterization of microbial communities. © 2024 Wiley Periodicals LLC. Basic Protocol: Microbial community profiling with Emu Support Protocol 1: Full-length 16S rRNA microbial sequences with Oxford Nanopore Technologies sequencing platform Support Protocol 2: Building a custom reference database for Emu.
摘要：
16SrRNA靶向扩增子测序是阐明微生物群落组成的既定标准。虽然高通量短读取测序由于其有限的读取长度而只能引发一部分16SrRNA基因，第三代测序可以完整读取16SrRNA基因，从而提供更精确的分类学分类。这里,我们提出了使用牛津纳米孔技术（ONT）生成全长16SrRNA序列的协议，以及使用Emu的微生物群落概况。我们选择Emu来分析ONT序列，因为它利用来自整个社区的信息来克服由于不完整的参考数据库和硬件限制而导致的错误，最终获得物种级别的分辨率。该管道提供了一种低成本的解决方案，通过利用实时，长读取ONT测序和定制的软件，用于准确表征微生物群落。©2024Wiley期刊有限责任公司。基本方案：使用Emu进行微生物群落分析支持方案1：使用OxfordNanoporeTechnologies测序平台进行全长16SrRNA微生物序列支持方案2：为Emu建立自定义参考数据库。
