I have so many sequences that I would like to classify to the “species” level with mothur . I was able to create OTUs to Genus level but couldn’t be able to do to species level? can someone explain me that? (which database file should I choose for example for 16s rRNA sequences?)
What commands did you run and with what parameters? When you say you couldn’t classify to the species level, what does the output for an OTU in your taxonomy file look like? And perhaps most importantly which database did you classify with? (SILVA database does not have species level information).
Remember that the classification is dependent on the database you are classifying against. Many organisms in the databases aren’t classified to species because we don’t know what species they are. Second, v4 may not contain enough information to classify to species even if closely related species are in the database-check out v4 for E. coli and Shigella for an example of this.
Some (including me) would argue that you’re asking too much to try and get to the species level using 250 nt from a 16S rRNA gene. This is why the databases generally do not include species level names. The greengenes database does provide some species, but only for about 10% of the genera. So that really isn’t very helpful. If you have a group that you really want to take to the species level, you would have to add multiple sequences per genus and multiple sequences per species and provide species-level taxonomy information for those sequences.
I agree that we can only go to the family or genus level if we only sequence ~ 250 bp fragment from the 16S rRNA gene. It seems that many researchers are interested in sequencing the DNA samples and going to the species level. Would you mind suggesting an easy-to-following protocol that we can use to get to the species level? thanks.