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

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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.
OriginalsprogEngelsk
Artikelnummer100437
TidsskriftCell Reports Methods
Vol/bind3
Udgave nummer3
Antal sider16
ISSN2667-2375
DOI
StatusUdgivet - 2023

Bibliografisk note

Funding Information:
This work was partially supported by a grant to Ou Wang from the National Natural Science Foundation of China ( 32001054 ). The samples were obtained from Dr. Yue-hua Hu, who is funded by the West Light Foundation of the Chinese Academy of Sciences.

Funding Information:
This work was partially supported by a grant to Ou Wang from the National Natural Science Foundation of China (32001054). The samples were obtained from Dr. Yue-hua Hu, who is funded by the West Light Foundation of the Chinese Academy of Sciences. F.F, O.W. and B.P. conceived the wet lab method. X.S. F.F. and O.W performed wet lab experiments and BGISEQ500 sequencing. C.F. and Z.S. conceived the bioinformatics method. C.F developed the software pipeline and performed data analysis as well as visualization. X.S. and X.Z. interpreted the microbial characteristics of data. X.S. performed the phylogenetic analysis. H.Z. Z.P. and X.L. assisted in performing the data. C.F. X.S. X.Z. O.W. B.P. Z.S. and K.K. wrote the manuscript. All authors participated in discussions and contributed to the revision of the manuscript. All authors read and approved the final manuscript. R.D. and B.P. were employed by the company Complete Genomics Inc. US.

Publisher Copyright:
© 2023 The Author(s)

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