If I understand right, you wonder about the result in the Blast search. You culture a sample from wastewater on a media, get some colonies growing, sequence them (probably 16S) and then Blast the result. Right?

There are different option how sequences can be entered to the database. If someone has a pure culture in use, which is described and known, the resulting sequences will always be affiliated to the species when an entry is done. These limited number of results you get by "excluding uncultured" in the Blast search options.

If you work with environmental samples, e.g. a soil sample or a wastewater treatment sample and you would like to know something about its diversity without culturing organisms, one option is to go for cloning and sequencing. Then you finally have a clone with a piece of your amplified 16S rDNA from the sample. The sequence you get is only an environmental sample. You can search for a group/species affiliation, but you will always enter it as environmental sample to the data base. Then it is usually entered as "uncultured species/organisms" because you never had the species as pure culture in your lab but only the isolated DNA from an environmental sample.

These envrionmental studies were quite common in the last years, so the data bases are full of these uncultured organsisms although there are also pure cutures of some of them available.

So if you aim to find the closest hit for described pure culture isolates for a given sequence you have to exclude the "uncultured" during your search. I you would like to get a general phylogenetic group of your colony I can also recommend the RDP classifier.

Please, be aware that culturing organisms from an environmental sample always just gives you a small number of species that can grow at the conditions you provide them. For wastewater this will be maximum 10 to 15 % of the species present in the sample. For general diversity analysis culturing is not the best option. But of course it always depends on your research question which approach you chose.

Hi Irma, could you describe your experimental intention a bit more detailed, please?

In general, when you would like to blast to get an idea about what you have cultured you can also exclude uncultures bacteria from the blast result (tick in search sheet).

Thank you Christin, I have two samples one direct from the environment and the other one from the plate, because is a wastewater treatment system and we have not many references about the species, and their ability to growth in the media we used, we explore the diversity. I understand the unculture results in the environmental sample but I can´t exclude this result in the blast from the plate samples because they are real and culturable.

If I understand right, you wonder about the result in the Blast search. You culture a sample from wastewater on a media, get some colonies growing, sequence them (probably 16S) and then Blast the result. Right?

There are different option how sequences can be entered to the database. If someone has a pure culture in use, which is described and known, the resulting sequences will always be affiliated to the species when an entry is done. These limited number of results you get by "excluding uncultured" in the Blast search options.

If you work with environmental samples, e.g. a soil sample or a wastewater treatment sample and you would like to know something about its diversity without culturing organisms, one option is to go for cloning and sequencing. Then you finally have a clone with a piece of your amplified 16S rDNA from the sample. The sequence you get is only an environmental sample. You can search for a group/species affiliation, but you will always enter it as environmental sample to the data base. Then it is usually entered as "uncultured species/organisms" because you never had the species as pure culture in your lab but only the isolated DNA from an environmental sample.

These envrionmental studies were quite common in the last years, so the data bases are full of these uncultured organsisms although there are also pure cutures of some of them available.

So if you aim to find the closest hit for described pure culture isolates for a given sequence you have to exclude the "uncultured" during your search. I you would like to get a general phylogenetic group of your colony I can also recommend the RDP classifier.

Please, be aware that culturing organisms from an environmental sample always just gives you a small number of species that can grow at the conditions you provide them. For wastewater this will be maximum 10 to 15 % of the species present in the sample. For general diversity analysis culturing is not the best option. But of course it always depends on your research question which approach you chose.