High-resolution single-molecule long-fragment rRNA gene amplicon sequencing of bacterial and eukaryotic microbial communities

作者：Chao Fang, Xiaohuan Sun, Fei Fan, Xiaowei Zhang, Ou Wang, Haotian Zheng, Zhuobing Peng, Xiaoqing Luo, Ao Chen, Wenwei Zhang, Radoje Drmanac, Brock A. Peters, Zewei Song, Karsten Kristiansen · 发表于：Cell Reports Methods · 年份：2023 · DOI：10.1016/j.crmeth.2023.100437 · 被引用次数：8 · 研究领域：Genomics and Phylogenetic Studies、Plant Pathogens and Fungal Diseases、Environmental DNA in Biodiversity Studies

Sequencing of hypervariable regions as well as internal transcribed spacer regions of ribosomal RNA genes (rDNA) is broadly used to identify bacteria and fungi, but taxonomic and phylogenetic resolution is hampered by insufficient sequencing length using high throughput, cost-efficient second-generation sequencing. We developed a method to obtain nearly full-length rDNA by assembling single DNA molecules combining DNA co-barcoding with single-tube long fragment read technology and second-generation sequencing. Benchmarking was performed using mock bacterial and fungal communities as well as two forest soil samples. All mock species rDNA were successfully recovered with identities above 99.5% compared to the reference sequences. From the soil samples we obtained good coverage with identification of more than 20,000 unknown species, as well as high abundance correlation between replicates. This approach provides a cost-effective method for obtaining extensive and accurate information on complex environmental microbial communities.