High-resolution single-molecule long-fragment rRNA gene amplicon sequencing of bacterial and eukaryotic microbial communities
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High-resolution single-molecule long-fragment rRNA gene amplicon sequencing of bacterial and eukaryotic microbial communities. / Fang, Chao; Sun, Xiaohuan; Fan, Fei; Zhang, Xiaowei; Wang, Ou; Zheng, Haotian; Peng, Zhuobing; Luo, Xiaoqing; Chen, Ao; Zhang, Wenwei; Drmanac, Radoje; Peters, Brock A.; Song, Zewei; Kristiansen, Karsten.
In: Cell Reports Methods, Vol. 3, No. 3, 100437, 2023.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - High-resolution single-molecule long-fragment rRNA gene amplicon sequencing of bacterial and eukaryotic microbial communities
AU - Fang, Chao
AU - Sun, Xiaohuan
AU - Fan, Fei
AU - Zhang, Xiaowei
AU - Wang, Ou
AU - Zheng, Haotian
AU - Peng, Zhuobing
AU - Luo, Xiaoqing
AU - Chen, Ao
AU - Zhang, Wenwei
AU - Drmanac, Radoje
AU - Peters, Brock A.
AU - Song, Zewei
AU - Kristiansen, Karsten
N1 - Publisher Copyright: © 2023 The Author(s)
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
KW - amplicon
KW - assembly
KW - bacteria
KW - co-barcoding
KW - cost-effective
KW - CP: microbiology
KW - fungi
KW - high-throughput
KW - rDNA
KW - sequencing
U2 - 10.1016/j.crmeth.2023.100437
DO - 10.1016/j.crmeth.2023.100437
M3 - Journal article
C2 - 37056375
AN - SCOPUS:85150520873
VL - 3
JO - Cell Reports Methods
JF - Cell Reports Methods
SN - 2667-2375
IS - 3
M1 - 100437
ER -
ID: 340545707