Most of ITS ASV's map to Other (>60%)

I did ITS sequencing of stool samples for Fungi. However when I look at relative abundances, even for phylum 60-70% of my relative abundance graph is Other DNA.

When I look at how my ASVs map with taxa from Unite Database, the majority of taxa do not map past Kingdom (only 27% of Phylum maps to a reference sequence, besides Kingdom:Fungi).

Is this normal? And is there anything I can do to filter the data so it's not just 90% other?

This is what was done already by a collaborator (Dada 1.16)

  • Amplicon sequence variants (ASVs): paired FASTQ reads were trimmed, and then filtered to remove reads containing Ns, or with maximum expected errors >=2.
  • Samples with fewer than 1,000 sequences were discarded. ASVs accounting for less than one millionth of all strain-level markers were discarded

Hi @kkl45,
I recommend BLASTing sequences that aren't getting assigned against NCBI nr to see what they hit. You can do that by cross-referencing the results of qiime feature-table tabulate-seqs, which will generate BLAST links for each ASV sequence, and qiime metadata tabulate on your FeatureData[Taxonomy] artifact, which will link the ASV IDs to their assigned taxonomy.

I suspect you might be picking up a lot of human reads, but this should help to sort that out.

EDIT: I created an issue to make this easier to assess in the future by optionally integrating FeatureData[Taxonomy] information in the tabulate-seqs output.

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