Rescript Plugin for 18S

[lmjackson]

Hi,
I have been using the Rescript plugin for classifer training using the Silva Reference Database to generate a taxa barplot for 18S data (following the thread here: [Processing, filtering, and evaluating the SILVA database (and other reference sequence data) with RESCRIPt]); however, I am still getting Bacterial sequence matches for some samples. Could this be a problem with the classifier training, or do I need to filter my results further to include only Eukaryotic results, or could this be due to low data quality? I have included only the taxonomic filtering script block below where I filtered to only include Eukaryota, is this the correct method for taxonomic restriction for classify 18S data?

qiime rescript filter-seqs-length-by-taxon
–i-sequences silva-138-ssu-nr99-seqs-cleaned.qza
–i-taxonomy silva-138-ssu-nr99-tax.qza
–p-labels Eukaryota
–p-min-lens 1400
–o-filtered-seqs silva-138-ssu-nr99-seqs-filt.qza
–o-discarded-seqs silva-138-ssu-nr99-seqs-discard.qza

[SoilRotifer (Mike Robeson)]

Hi @lmjackson, welcome to !

@lmjackson

however, I am still getting Bacterial sequence matches for some samples.

Can you provide details on which primer set you used to generate your sequence data? Many primer sets will amplify off-target sequences. That is just the nature of the process. How much of your sequence data is being returned as Bacterial? Few? Many?

@lmjackson

Could this be a problem with the classifier training, or do I need to filter my results further to include only Eukaryotic results, or could this be due to low data quality? I have included only the taxonomic filtering script block below where I filtered to only include Eukaryota, is this the correct method for taxonomic restriction for classify 18S data?

I would advise against removing removing groups of taxa just because your are unable to identify what you want. In fact, it is best to leave these "outgroup" taxa within your database. This way you can more confidently remove off-target sequence data. Otherwise, you may falsely classify many sequences as simply Eukaryotes, when in fact they are not.

What sequencing protocol was used? That is, is your sequence data in mixed orientation? This can fool you into thinking your data are composed of bacterial sequences. See the following forum threads:

Import custom taxonomy

Hi @jose_gacia, What command did you run when trying to classify your reads with SILVA? When we observe many poor taxonomy assignments like this, it is typically due to mixed read orientation. I’d recommend using vsearch as outlined here: Or installing the RESCRIPt plugin, and running rescript orient-seqs on your reads. Then retry the BLAST and/or naíve-bayes classifiers. -Mike

50% unassigned sequences after taxonomy

Welcome @Lu_Wang! From your description, it sounds like you are aware that the mixed orientation of your reads (mix of forward and reverse sequences) is causing the 50% unassigned rate. This is because the classify-sklearn method can only handle reads in a single orientation. They can be forward or reverse relative to the reference, but all query sequences must be oriented. If your sequences are in mixed orientations, you can do one of two things: use the classify-consensus-vsearch method …

-Cheers!

[lmjackson]

Hi @SoilRotifer,

Thank you for your quick response!

I am using the E572F and E1009R primer set to generate the sequence data, and one entire sample was returned as 100% bacterial. These samples were sent to me by a colleague, but I am guessing that they could be of mixed orientation. After reading the forum threads you suggested, I will try using the orient-seqs method and re-running the classifier to see if this helps.

Also, thanks for the helpful suggestion to not filter out specific taxonomic groups because of the potential for having false classifications, I will be sure to retain these outgroup taxa within my database in the future.