Sequences disappeared after running qiime feature-table merge-seqs

Technical Support
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1

Hi Team,
I'm experiencing similar/same issue as mentioned in topic : sequences disappear with this command: qiime feature-table merge-seqs
which wasn't resolved. After merging several representative_sequences.qza artifacts it turned out that 2 of the sequences disappeared in the merged file - the features are present in merged table artifact and the way I discovered it was after getting error message when trying to run qiime taxa barplot fot the merged feature table with taxonomy created from merged representative_sequences.qza artifact.
My commands were:

qiime feature-table merge --i-tables /media/storage/Galanova_0520/H4/dada2out/table.qza   /media/storage/Galanova_0520/H5/dada2out/table.qza  /media/storage/Galanova_0520/H6/dada2out/table.qza   --o-merged-table /media/storage/Galanova_0520/Fungi_joined/merged_table  --p-overlap-method sum;
qiime feature-table summarize --i-table /media/storage/Galanova_0520/Fungi_joined/merged_table.qza  --m-sample-metadata-file /media/storage/Galanova_0520/Fungi_all_mapping.txt  --o-visualization /media/storage/Galanova_0520/Fungi_joined/merged_table;
qiime feature-table merge-seqs --i-data  /media/storage/Galanova_0520/H4/dada2out/representative_sequences_H4.qza   /media/storage/Galanova_0520/H5/dada2out/representative_sequences_H5.qza   /media/storage/Galanova_0520/H6/dada2out/representative_sequences_H6.qza    --o-merged-data  /media/storage/Galanova_0520/Fungi_joined/rep-seqs-dada2_full_merged;
qiime feature-table tabulate-seqs --i-data  /media/storage/Galanova_0520/Fungi_joined/rep-seqs-dada2_full_merged.qza  --o-visualization /media/storage/Galanova_0520/Fungi_joined/rep-seqs-dada2_full_merged;

I'm running qiime2-2021.4 nativelly installed.
Thanks for any advice.
Martin

2

Hi @Martin_Kostovcik - can you please share the two summary QZVs you listed above:

  1. merged_table.qzv
  2. rep-seqs-dada2_full_merged.qzv
3

Hi,
here it is. Thanks for looking into it and eny clues about the issue.
Martinrep-seqs-dada2_full_merged[1].qzv (523.0 KB) merged_table[1].qzv (572.2 KB)

5

Thanks for sharing those files @Martin_Kostovcik!

Can you elaborate further on why you think you're missing features? The two visualizations you shared indicate that the feature count is matched between the two:

merged seqs:

Screen Shot 2021-06-30 at 11.39.59 AM
Screen Shot 2021-06-30 at 11.39.59 AM1608×356 30.7 KB

merged tables:

Screen Shot 2021-06-30 at 11.40.08 AM
Screen Shot 2021-06-30 at 11.40.08 AM1666×424 24.2 KB

7

Oooh,
my fault. Sorry I was a bit confused with the identical name of the file and uploaded a different on. So once again and better attaching two rep_seq files which after merging lost 2 features - fc57242f57cf161639b4180ff801742b and 355f5cde4f0b2ac57f32bb419ff55c33. I found out that after getting error from classification abous missing features. Those features are present in the first rep_seq files being merged.....no idea what happened. Sorry once again for attaching incorrect file i was in hurry a bit.
The command was : qiime feature-table merge-seqs --i-data /media/storage/Galanova_0520/Fungi_joined/rep-seqs-dada2_full_merged.qza /media/storage/Kostovcikova_bobky_0521/210510_Kostovcikova20210510/raw_fastq/Fungi/KostH4/dada2out/representative_sequences.qza --o-merged-data /media/storage/Kostovcikova_bobky_0521/210510_Kostovcikova20210510/raw_fastq/Fungi/TD_KK_OLOMOUC_FUNG_JOINED_W_0520/rep-seqs-dada2_full_merged;
rep-seqs-dada2_full_merged[1].qzv (523.0 KB) rep-seqs-dada2_full_merged[1].qzv (683.3 KB)
The second one is the merged file.
Thanks for any clue.
Martin

8

Hi @Martin_Kostovcik - I suggest you double-check your book-keeping - these two visualizations have completely different provenance. If you trace back, even the import steps show that you imported different data between the two runs. Double-check that you have everything in order and let us know what you find.

9

Hi Matt,
so I double-checked the files renamed the outputs and it's working now by what I mean those two missing features are in the merged table as well as in merged rep_seq file. I used exactly the same command as in a previous post just changed the name of the output file. I'm attaching once again both files - the old one with missing features (fc57242f57cf161639b4180ff801742b; 355f5cde4f0b2ac57f32bb419ff55c33) and the new one without missing features. Both files as I mentioned, were created using the same command : qiime feature-table merge-seqs --i-data /media/storage/Galanova_0520/Fungi_joined/rep-seqs-dada2_full_merged.qza /media/storage/Kostovcikova_bobky_0521/210510_Kostovcikova20210510/raw_fastq/Fungi/KostH4/dada2out/representative_sequences.qza --o-merged-data /media/storage/Kostovcikova_bobky_0521/210510_Kostovcikova20210510/raw_fastq/Fungi/TD_KK_OLOMOUC_FUNG_JOINED_W_0520/rep-seqs-dada2_full_merged_test; There is a difference of 5 extra features in the latter "test" file.rep-seqs-dada2_full_merged[1].qzv (683.3 KB) rep-seqs-dada2_full_merged_test[1].qzv (686.5 KB)
No idea where was a problem but it's working now. Thanks for your help and guidance.

11

Hi @Martin_Kostovcik - the initial data import represented completely different data (I'll include just the md5 sums for the barcode files imported, for brevity)

  • viz #1 (UUID 193291cf-6b93-4f63-82bd-72d11f47f632):
    • 7e4716b1a417ab41344a7c30dd4dd5df
    • 9dca27c5264f9651a1f6be5dc3b2692a
    • a84ed9453e5a263ef12da9fc8de1b37c
  • viz #2 (UUID 416e832d-c25b-4b50-8645-61af65d31f3a) (note, this file was shared twice in this thread):
    • 26f10d401ea1f3d1791b5b1a6e513775
    • 442d55f690a8ce50fd91a23c98a37595
    • 8d4a528bedb4426a364d5de895bbb0d1
    • f0c2f5321f4451ffd3efa29bf425836c
  • viz #3 (UUID c5d9035f-5774-4970-976c-8f5074154880)
    • 7e4716b1a417ab41344a7c30dd4dd5df
    • 8d4a528bedb4426a364d5de895bbb0d1
    • 9dca27c5264f9651a1f6be5dc3b2692a
    • a84ed9453e5a263ef12da9fc8de1b37c

This is why I'm thinking you have a book-keeping issue - it sounds like you got everything sorted out, but in case something like this happens again, pay careful attention to what files you are using, and where/when. Good luck!

:qiime2:

PS - you can see those md5sums here when you click on the "provenance" tab on view.qiime2.org after loading a file:

Screen Shot 2021-07-27 at 12.23.13 PM

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