incomplete taxonomic assignment

[SoilRotifer (Mike Robeson)]

@Saptarathi_Deb

It consists of 6 amplicons of length 200-250 base pair covering 7 variable regions

This is not much of a problem...

@Saptarathi_Deb

One issue is that the primers are unknown. The way we are dealing is by 1) trimming 20 base pairs from both sides. OR 2) Crack the primer sequence to the extent possible and use cut adapt to trim them (however, the reads are of mixed orientation from the sequencing machine, so it's less efficient sometimes I feel).

• They are using proprietary primers? Often companies are willing to tell you how many bases to trim. Did you ask them for these details?
• Are you sure the primers are even in the resulting sequence data? I ask because not all protocols sequence "through" the primer.
• Mixed orientation:
  • Trimming primers from mixed orientated reads is not a problem for cutadapt.
  • You can also simply re-orient all of your reads to a reference database (e.g. SILVA ), using the orient-seqs command from the RESCRIPt plugin.

@Saptarathi_Deb

Since I want the classification to happen without separating the reads based on variable regions, it's not possible to use classify-sklearn.

This is not true. You can simply use the full-length SILVA classifier for all variable regions.

@Saptarathi_Deb

I am trying to use classify-sklearn now by separating the reads region by region. I hope it works well.

This will work too of course. But you might be better off with my next suggestion.

@Saptarathi_Deb

The only reason I am a little greedy to classify them to genus level is that I realized some of the partially classify reads could change the differential abundance statistics if we take them re-classify using vsearch or NCBI BLAST, and add them to their original taxa.

Have you looked into the q2-sidle plugin? You'll be able to leverage all of your amplicon regions to obtain a better taxonomy.

-Cheers!
-Mike