I did 16srRNA sequencing (sangar platform) of PCR amplicons. Did trimming of forward and reverse chromatogram reads and merged them using HVDR Fragment Merger Tool using different parameters. With each parameter, I am getting a different BLAST result. Although the genus bacillus is the same at the species level. But sometimes it shows paralicheniformis, haynesii, piscis, sonorensis, and licheniformis. The Percentage, query coverage, and E value are exactly the same. It seems to be a conserved sequence but how can I proceed to identify it to species level.
* With each parameter, I am getting a different BLAST result.
>> This is not surprising, if you are changing the parameter, you can get different results. Either do not change any parameter, or keep the parameter same for all your analyses.
* Although the genus bacillus is the same at the species level.
>> This is not the correct way how one should form a sentence like this. Better way would be, all species belong to genus Bacillus.
* he Percentage, query coverage, and E value are exactly the same. It seems to be a conserved sequence but how can I proceed to identify it to species level.
>> For genus like Bacillus, 16S alone simply is not a good marker for the identification at species level. Use Other markers in addition to the 16S gene.
Ahmer Bin Hafeez
Check the following:
- Marston, C.K., Gee, J.E., Popovic, T. et al. Molecular approaches to identify and differentiate Bacillus anthracis from phenotypically similar Bacillus species isolates. BMC Microbiol 6, 22 (2006). https://doi.org/10.1186/1471-2180-6-22
- https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/697260/ID_9i3.1.pdf
you should do a full genome sequencing to be more precise, then you build a phylogenetic tree with the closest species given to you by RNA16s Blast.
Best luck !
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