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Method Article
Janet E. Hill
University of Saskatchewan
Corresponding Author
ORCiD: https://orcid.org/0000-0002-2187-6277
License:
This work is licensed under a CC BY 4.0 License. Read Full License
This protocol can be applied to determine the composition of a microbial community. The cpn60 gene (also known as groEL, hsp60) is present in almost all bacteria and a 552-558 bp region of the gene has been established as a barcode for species level identification of bacteria. The primer cocktail used in this protocol amplifies cpn60 barcode sequences from bacteria with a wide range of G+C content. Some species of Mycoplasma lack the cpn60 gene and therefore this method is not recommended to detect Mycoplasma. DNA sequences generated from this method could be compared to cpnDB, a public database of cpn60 sequences, for identification. Library preparation involves cpn60 amplicon generation, PCR clean-up, index PCR, index PCR clean-up, library quantification, normalization, pooling, library denaturation and loading. Time taken to complete depends on the number of samples included. If using 96 samples, the procedure takes 8 hours but there are several stages where the samples could be stored and continued the next day. Specific instructions are provided for the Illumina MiSeq platform, but the protocol could easily be adapted for other sequencing platforms.
Keywords
metagenomics, microbial ecology, Miseq, cpn60, barcode, amplicon sequencing, PCR, microbiome, marker gene
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Posted 30 Mar, 2021
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An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Janet E. Hill
University of Saskatchewan
Corresponding Author
ORCiD: https://orcid.org/0000-0002-2187-6277
Version History
CURRENT STATUS: Posted
Version 1
Posted 30 Mar, 2021
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
This protocol can be applied to determine the composition of a microbial community. The cpn60 gene (also known as groEL, hsp60) is present in almost all bacteria and a 552-558 bp region of the gene has been established as a barcode for species level identification of bacteria. The primer cocktail used in this protocol amplifies cpn60 barcode sequences from bacteria with a wide range of G+C content. Some species of Mycoplasma lack the cpn60 gene and therefore this method is not recommended to detect Mycoplasma. DNA sequences generated from this method could be compared to cpnDB, a public database of cpn60 sequences, for identification. Library preparation involves cpn60 amplicon generation, PCR clean-up, index PCR, index PCR clean-up, library quantification, normalization, pooling, library denaturation and loading. Time taken to complete depends on the number of samples included. If using 96 samples, the procedure takes 8 hours but there are several stages where the samples could be stored and continued the next day. Specific instructions are provided for the Illumina MiSeq platform, but the protocol could easily be adapted for other sequencing platforms.
Figure 1
Figure 2
Figure 3
Figure 4
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