Analyzing whole-metagenomic shotgun sequencing data with V4 amplicon sequence data

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@jwdebelius

Let me tell you the whole scenario. I've downloaded sequence data from the SRA database with the accession number PRJNA672260 to test the q2-greengenes plugins. These sequences are 16S rRNA V3-V4 regions. So first I have run the following code using deblur to produce 150nt long sequences.

qiime quality-filter q-score \
  --i-demux paired_reads.qza \
  --o-filtered-sequences demux-filtered_PRJNA672260.qza \
  --o-filter-stats demux-filter-stats_PRJNA672260.qza
qiime deblur denoise-16S \
  --i-demultiplexed-seqs demux-filtered_PRJNA672260.qza \
  --p-trim-length 150 \
  --p-sample-stats \
  --o-representative-sequences rep-seqs-deblur_PRJNA672260_150.qza \
  --o-table table-deblur_PRJNA672260_150.qza \
  --o-stats deblur-stats_PRJNA672260_150.qza

Here is the files if you want to look into it,
deblur-stats_PRJNA672260_150.qza (33.4 KB)
rep-seqs-deblur_PRJNA672260_150.qza (102.5 KB)
table-deblur_PRJNA672260_150.qza (162.3 KB)
and their visualization files,
table-deblur_PRJNA672260_150.qzv (507.9 KB)
paired_reads.qzv (328.1 KB)
metadata.qzv (1.2 MB)

Then I considered table-deblur_PRJNA672260_150.qza file for the next q2-greengenes2 pipeline. Then I tried to follow the following instructions and write my code as follows,

qiime greengenes2 filter-features \
  --i-feature-table table-deblur_PRJNA672260_150.qza \
  --i-reference 2022.10.taxonomy.asv.nwk.qza \
  --o-filtered-feature-table table-deblur_PRJNA672260_150_gg2.qza
qiime greengenes2 taxonomy-from-table \
  --i-reference-taxonomy 2022.10.taxonomy.asv.nwk.qza \
  --i-table table-deblur_PRJNA672260_150_gg2.qza \
  --o-classification table-deblur_PRJNA672260_150_sequence_gg2.qza

But this shows an error report like this,

Traceback (most recent call last):
File "/home/turtle/miniforge3/envs/qiime2-2023.7/lib/python3.8/site-packages/q2cli/commands.py", line 478, in call
results = self._execute_action(
File "/home/turtle/miniforge3/envs/qiime2-2023.7/lib/python3.8/site-packages/q2cli/commands.py", line 539, in _execute_action
results = action(**arguments)
File "", line 2, in taxonomy_from_table
File "/home/turtle/miniforge3/envs/qiime2-2023.7/lib/python3.8/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
outputs = self.callable_executor(
File "/home/turtle/miniforge3/envs/qiime2-2023.7/lib/python3.8/site-packages/qiime2/sdk/action.py", line 566, in callable_executor
output_views = self._callable(**view_args)
File "/home/turtle/miniforge3/envs/qiime2-2023.7/lib/python3.8/site-packages/q2_gg2/_methods.py", line 778, in taxonomy_from_table
tree = _load_tree_and_cache(open(str(reference_taxonomy)), features)
File "/home/turtle/miniforge3/envs/qiime2-2023.7/lib/python3.8/site-packages/q2_gg2/_methods.py", line 543, in _load_tree_and_cache
tree = tree.shear(names & features)
File "bp/_bp.pyx", line 758, in bp._bp.BP.shear
File "bp/_bp.pyx", line 800, in bp._bp.BP.shear
ValueError: No requested tips found

what I've managed to learn is that after the referencing with 2022.10.taxonomy.asv.nwk.qza file the resulting table-deblur_PRJNA672260_150_sequence_gg2.qza file lists all the samples in it.
table-deblur_PRJNA672260_150_gg2.qzv (394.5 KB)

So, can you now suggest me any solution? or why I'm having this much trouble? If you can't help me is there anyone with an understanding of this problem?

1 Like