I did 16S PCR and Sanger sequencing to see if the expected bacteria were present in my co-culture experiments. I did a nucleotide BLAST under the refseq_genomic database for highly similar sequences. The BLAST with a genus type specified showed results demonstrating the presence of the expected bacteria in my experiments. However, the refseq_genomic BLAST without a genus specified did not. Why is there a difference between the two? Should i only care about one or the other?
Yes there will be different result in case of divergent between query sequence and the specified genus. But if there is close relationship between genus and the query then both BLASTs will give similar result.
In your case, 16S RNA sequence of sequenced strain may have large divergent to your culture. I think It may different a strain.
Although BLAST is a powerfull tool for many things, when trying to classify bacteria through 16S sequencing, I would rather use the RDP classifier:
https://rdp.cme.msu.edu/classifier/classifier.jsp
It is a specialized database that contains mostly bacterial 16S sequences (they also have more and more archae and fungi ITS) with the corresponding classification, they curate and update it regularly to make sure it is correct, and their classifier has a bootstrap algorithm that gives you percentage of confidence of the classification for each depth of the phylogeny.
In my experience, RDP is better for bacteria classification, although the first few hits in BLAST should point you in the same direction as RDP.
Maybe you can try it and let us know what the result is?
Hi I used RDP on the sequence and it gave me Prevotella (which is what is expected and same for ncbi BLAST) for genus, but nothing for species.
Hi, Sorry I didn't get that you wanted to have species-level classification. In general it is not advised to try to classify bacteria to species-level precision only using the 16S gene (even full length). That's why RDP will only give you classification to genus-level, because it is not possible to reach a good enough confidence to attribute to species (I believe they explain it on the RDP website). It is the same with BLAST, you should not take the best hit and conclude that your sequence belong to the same species. You can take it as a hint but nothing more (you can also look in RDP which sequences looked more similar to yours and see which species they belong to, but again, it's only a hint). If you really want to precisely identify a bacterial species you have to go with another method than 16S sequencing, for example whole genome sequencing, but this method is also limited as not many bacteria species genomes are available in the databases (the majority of the genomes belong to E.coli), so depending on your bacteria of interest, you might not get much genomes to compare it with.
Marie Vallier's answers are correct! I would just like to complement saying that although the gold standard for identifying bacteria is 16S rRNA, other genes can be used as rpoB, as well as techniques that do not require complete sequencing like MLST (Multilocus sequence typing-more informations about here: http://www.mlst.net/misc/further.asp).
When you want to identify bacteria at the species level you will need to use at least MLST or sequencing the entire genome, the 16S rRNA gene gives only sufficiently good results to genere. Also in any identification technique it is always good to search results in different databases to confirm the results.
In conclusion, you may see somewhat different results when you restrict the BLAST search, because limiting the BLAST database will have a smaller dataset to look for similarities.
- Badges
- Science method