# How can I classify OTUs to "SPECIES" level with mothur? **URL:** \ **Category:** Theory behind mothur\ **Created:** [May 9, 2016, 8:59am UTC](https://forum.mothur.org/t/how-can-i-classify-otus-to-species-level-with-mothur/2837 "2016-05-09T08:59:36Z")\ **Posts on this page:** 10\ **Page:** 1
**Author:** ![mhmtgenc](https://avatars.discourse-cdn.com/v4/letter/m/9fc348/32.png) [@mhmtgenc](https://forum.mothur.org/u/mhmtgenc)\ **Post date:** [May 9, 2016, 8:59am UTC](https://forum.mothur.org/t/how-can-i-classify-otus-to-species-level-with-mothur/2837/1 "2016-05-09T08:59:36Z")
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?) Please help me I am getting pretty lost 🙂 ---
**Author:** ![campenr](https://avatars.discourse-cdn.com/v4/letter/c/8dc957/32.png) [@campenr](https://forum.mothur.org/u/campenr)\ **Post date:** [May 9, 2016, 3:17pm UTC](https://forum.mothur.org/t/how-can-i-classify-otus-to-species-level-with-mothur/2837/2 "2016-05-09T15:17:55Z")
Can you provide more information? 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). ---
**Author:** ![Kendra](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/kendra/32/112_2.png) [@Kendra](https://forum.mothur.org/u/Kendra)\ **Post date:** [May 9, 2016, 6:33pm UTC](https://forum.mothur.org/t/how-can-i-classify-otus-to-species-level-with-mothur/2837/3 "2016-05-09T18:33:01Z")
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. ---
**Author:** ![mhmtgenc](https://avatars.discourse-cdn.com/v4/letter/m/9fc348/32.png) [@mhmtgenc](https://forum.mothur.org/u/mhmtgenc)\ **Post date:** [May 10, 2016, 6:22am UTC](https://forum.mothur.org/t/how-can-i-classify-otus-to-species-level-with-mothur/2837/4 "2016-05-10T06:22:02Z")
> [@campenr](#): > > Can you provide more information? > > 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). Thanks for the reply and here is my output; “OTU Size Taxonomy Otu00001 25137 Bacteria(100) Proteobacteria(100) Betaproteobacteria(100) Burkholderiales(100) Alcaligenaceae(100) Bordetella(99) Otu00002 15182 Bacteria(100) Proteobacteria(100) Alphaproteobacteria(100) Rhodobacterales(100) Rhodobacteraceae(100) Paracoccus(100) Otu00003 2758 Bacteria(100) Proteobacteria(100) Betaproteobacteria(100) Burkholderiales(100) Alcaligenaceae(100) Bordetella(99) Otu00004 2493 Bacteria(100) Proteobacteria(100) Alphaproteobacteria(100) Rhodobacterales(100) Rhodobacteraceae(100) Paracoccus(100) Otu00005 1516 Bacteria(100) Proteobacteria(100) Betaproteobacteria(100) Burkholderiales(100) Alcaligenaceae(100) Bordetella(77) Otu00006 1436 Bacteria(100) Proteobacteria(100) Alphaproteobacteria(61) Rhodobacterales(52) Rhodobacteraceae(52) unclassified(100)” And can’t be sure which database I should use? > [@kmitchell](#): > > 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. And thank you too my friend and again my problem is not knowing how to choose a database and how to modify it please help on his? ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [May 10, 2016, 12:15pm UTC](https://forum.mothur.org/t/how-can-i-classify-otus-to-species-level-with-mothur/2837/5 "2016-05-10T12:15:46Z")
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. Pat ---
**Author:** ![thedam](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/thedam/32/79_2.png) [@thedam](https://forum.mothur.org/u/thedam)\ **Post date:** [November 21, 2019, 4:36pm UTC](https://forum.mothur.org/t/how-can-i-classify-otus-to-species-level-with-mothur/2837/6 "2019-11-21T16:36:12Z")
here they claim it’s possible for 14 species vs Silva v123 [https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176555](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176555) ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [November 21, 2019, 6:55pm UTC](https://forum.mothur.org/t/how-can-i-classify-otus-to-species-level-with-mothur/2837/7 "2019-11-21T18:55:39Z")
That paper is wrong. They got way out over their skis on the claims they made in there. ---
**Author:** ![Kendra](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/kendra/32/112_2.png) [@Kendra](https://forum.mothur.org/u/Kendra)\ **Post date:** [November 22, 2019, 4:07pm UTC](https://forum.mothur.org/t/how-can-i-classify-otus-to-species-level-with-mothur/2837/8 "2019-11-22T16:07:27Z")
⛷⛷⛷ so pleased there’s an emoji for this ---
**Author:** ![ZhangChiqian](https://avatars.discourse-cdn.com/v4/letter/z/94ad74/32.png) [@ZhangChiqian](https://forum.mothur.org/u/ZhangChiqian)\ **Post date:** [December 12, 2019, 8:15pm UTC](https://forum.mothur.org/t/how-can-i-classify-otus-to-species-level-with-mothur/2837/9 "2019-12-12T20:15:02Z")
> [@pschloss](#): > > sking too much to try and get to the species level using 250 nt from a 16S rRNA gene 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. ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [December 12, 2019, 8:46pm UTC](https://forum.mothur.org/t/how-can-i-classify-otus-to-species-level-with-mothur/2837/10 "2019-12-12T20:46:47Z")
You would need to sequence it’s genome