12S Taxonomic Assignment with Ref Genome

[lastewart (Lisa_Stew)]

Hi,

I need to run taxonomic assignment for some 12S data for birds, but I do not have a good reference database that includes my species of interest. I do have some reference genomes, but I was wondering if this is good practice to use, since it will essentially be forcing assignment on a single species reference sequences. I didn't see anything in the forum about using un-annotated reference genomes for target species without reference sequences, and 12S sequences for birds are not as well represented as MiFish. What is the best approach for assigning taxonomy in this case?

[Nicholas_Bokulich]

Hi @lastewart ,
Welcome to the forum!

You could check NCBI GenBank for 12S seqs for birds. If it is sufficient enough, then RESCRIPt would be a good solution. See some examples here for making (non-bird) 12S databases with RESCRIPt:

Make 12S reference database using Rescript

Hi @Junli_Zhang, I’ve got quite a bit of experience in making 12S rRNA gene databases (e.g. for eukaryota, and metazoa). Hopefully, we can help. I agree with @Nicholas_Bokulich, there are things beyond our control when it comes to internet connections. Your query does not appear to result in that many sequences, but you can break it up into smaller downloadable chunks within the vertebrates. Here is a query you can use to download Gnathostomata: txid7776[ORGN] AND (12S OR 12S…

Filter Sequences by Length BEFORE Downloading with get-ncbi-data

Hi there, I've been using and loving rescript for my eDNA metabarcoding projects. I was wondering if it was possible to filter sequences returned from NCBI before downloading them. For example, I'm doing a metabarcoding project using the 12S gene targeting fishes. One of the target species Percina brevicauda (txid163812) has its entire mitochondrial genome sequenced, but does not have individual entries for just the 12S gene. If I were to use this search, I would not get any results: qiime re…

You can check GenBank first to test out a query and see how many hits you get.

Good luck!

[lastewart (Lisa_Stew)]

@Nicholas_Bokulich

Thanks for the quick response and the helpful links!
I will try out the suggested methods for NCBI extraction and RESCRIPT, hopefully I will have some luck with that. I think the one issue with these methods is that there aren't annotated gene sequences for the target or related species I am identifying, only a few recent whole reference genomes not yet on NCBI. Is there an alternative way to use reference genomes to identify whether a species is present or not within a sample? Sorry if this may just be a basic blast query solution, I am fairly new to this field.

[Nicholas_Bokulich]

@lastewart

Is there an alternative way to use reference genomes to identify whether a species is present or not within a sample?

The best thing to do would be to trim the 12S sequences from the full genomes. That part would need to be done outside of QIIME 2, as the current sequence trimmer is not designed for trimming multi-copy genes from whole genome sequences.

Once you have a FASTA of 12S sequences, these could be imported into QIIME 2 as a FeatureData[Sequence] artifact and used as your reference database.

Good luck!