Improvement in Microbiota Recovery Using Cas-9 Digestion of Mānuka Plastid and Mitochondrial DNA.
16S rRNA gene amplicon sequencing
Cas-16S-seq
Host contamination
Microbiota profiling
Plant microbiome
Journal
Microbial ecology
ISSN: 1432-184X
Titre abrégé: Microb Ecol
Pays: United States
ID NLM: 7500663
Informations de publication
Date de publication:
09 Oct 2024
09 Oct 2024
Historique:
received:
06
05
2024
accepted:
19
09
2024
medline:
9
10
2024
pubmed:
9
10
2024
entrez:
8
10
2024
Statut:
epublish
Résumé
Understanding host-microbe interactions in planta is an expanding area of research. Amplicon sequencing of the 16S rRNA gene is a powerful and common method to study bacterial communities associated with plants. However, the co-amplification of mitochondrial and plastid 16S rRNA genes by universal primers impairs the sensitivity and performance of 16S rRNA sequencing. In 2020, a new method, Cas-16S-seq, was reported in the literature to remove host contamination for profiling the microbiota in rice, a well-studied domestic plant, by engineering RNA-programmable Cas9 nuclease in 16S rRNA sequencing. For the first time, we tested the efficiency and applicability of the Cas-16S-seq method on foliage, flowers, and seed of a non-domesticated wild plant for which there is limited genomic information, Leptospermum scoparium (mānuka). Our study demonstrated the efficiency of the Cas-16S-seq method for L. scoparium in removing host contamination in V4-16S amplicons. An increase of 46% in bacterial sequences was found using six guide RNAs (gRNAs), three gRNAs targeting the mitochondrial sequence, and three gRNAs targeting the chloroplast sequence of L. scoparium in the same reaction. An increase of 72% in bacterial sequences was obtained by targeting the mitochondrial and chloroplast sequences of L. scoparium in the same sample at two different steps of the library preparation (DNA and 1st step PCR). The number of OTUs (operational taxonomic units) retrieved from soil samples was consistent when using the different methods (Cas-16S-seq and 16S-seq) indicating that the Cas-16S-seq implemented for L. scoparium did not introduce bias to microbiota profiling. Our findings provide a valuable tool for future studies investigating the bacterial microbiota of L. scoparium in addition to evaluating an important tool in the plant microbiota research on other non-domesticated wild species.
Identifiants
pubmed: 39379709
doi: 10.1007/s00248-024-02436-6
pii: 10.1007/s00248-024-02436-6
doi:
Substances chimiques
RNA, Ribosomal, 16S
0
DNA, Mitochondrial
0
DNA, Bacterial
0
RNA, Guide, CRISPR-Cas Systems
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
124Subventions
Organisme : Ministry of Business, Innovation and Employment
ID : C11X1803
Organisme : Ministry of Business, Innovation and Employment
ID : C11X1803
Organisme : Ministry of Business, Innovation and Employment
ID : C11X1803
Organisme : Ministry of Business, Innovation and Employment
ID : C11X1803
Informations de copyright
© 2024. The Author(s).
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