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16S rRNA gene and ITS region amplification, sequencing, and phylogenetic analysis

The amplification and sequencing of the 16S rRNA gene is a routine method used nowadays to help with the identification and classification of Cyanobacteria as part of a polyphasic approach workflow. An unknown isolate can be identified by comparing the similarity of its 16S rRNA sequence with 16S rRNA sequences of strains with known taxonomic identity, which are currently present in public databases. The ITS region, located between the 16S and 23S rRNA genes, is also used nowadays in cyanobacterial taxonomy. Due to its high variability, it allows a better taxonomic resolution than the 16S rRNA gene alone and can be used to support the findings of 16S rRNA phylogenetic analyses.

The service can be offered for 16S rRNA or ITS region alone or both (16S rRNA and ITS), depending on customer’s needs.

 

Workflow

We carry out the amplification of 16S rRNA gene followed by Sanger sequencing, which results in a quasi-complete nucleotide sequence of 16S rRNA gene and a complete nucleotide sequence of ITS region. Certain cyanobacteria possess multiple copies of their ribosomal operon. To overcome this issue, bands are gel-purified prior to sequencing. The raw sequencing data are inspected, edited, and consensus sequences are obtained. All consensus sequences are checked for chimeras prior to phylogenetic analysis.

The analysis report provided to our customers includes a description of the used methodology, a list of the most closely related species available in the GenBank database at the time of the analysis, with the obtained sequence similarities, a phylogenetic tree (ML and/or BI) for each analyzed gene marker and a 16S rRNA similarity matrix (based on p-distance). For ITS region, conserved domains are also identified and folded. Finally, the report is accompanied by a scientific conclusion on the identity of the provided strain(s).

It is noted that safe conclusions related to the taxonomic placement of each strain can be drawn only by combining the obtained molecular data with a thorough morphological assessment.

 

For this analysis, a unicyanobacterial culture in good shape is required.

Please contact us for more information or if you wish to order this analysis.