NameError: name 'DataSecondALL' is not defined. while running puppy-primers -p group

[xmeng]

Hello,

Thanks for developing the tool. I installed puppy 1.0.3 and am able to run puppy-align on a community of 120 microbes. I am able to get all files generated correctly. However, I got NameError: name 'DataSecondALL' is not defined. while running puppy-primers -p group on a selected group of 3 microbes. Any clues to fix the error? Thanks in advance. Xmeng

available puppy-align files: primer3_files Stats_pipelineOutput.tsv UniqueGenesList.tsv UniqueGenesPlot.pdf UniquePrimerTable.tsv

puppy version: puppy 1.0.3 py310_0 hghezzi

cmd: puppy-primers -p group -pr group_input -i align_out/ResultDB.tsv -o primer_group_out3

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ASCII art designed with manytools.org from puppy logo

Renaming FASTA headers (if needed)...

Loading the alignments file (align_out/ResultDB.tsv, size = 1.298060774 GB) as a Dask Dataframe... Converting Dask Dataframe to Pandas dataframe... No ideal group genes identified that amplify all the target organisms. Could not find "second choice" candidate group genes for the target organisms Skipping parsing on optimal genes. Skipping parsing on suboptimal genes. Optimal genes not found. Won't ceate file. Subotimal genes not found. Won't ceate file. Beginning to design group-specific primers for the target organisms provided... Traceback (most recent call last): File "/home/ec2-user/miniconda3/envs/puppy/bin/puppy-primers", line 1201, in elif (len(DataIdeal) == 0) and (len(DataSecondALL) > 0): # Design group primers using 'second choice' genes NameError: name 'DataSecondALL' is not defined. Did you mean: 'DataSecond'?

[hghezzi]

Hi!

Thank you for reaching out and providing the error message :)

It is difficult to replicate because of the 120 files, but it seems like puppy could not identify any genes that would selectively amplify your 3 members in the community of 120. The text Beginning to design group-specific primers for the target organisms provided... seems to be a bug on our end, as based on the previous messages puppy should not be attempting to design any primers due to the lack of good candidate genes. I will fix that in the new version once ready to be realised.

In the meanwhile, can you see the files "IdealGroupGenes.tsv" and/or "SecondChoiceGroupGenes.tsv" in your primer_group_out3 folder? Are they empty?

Are your 3 organisms closely related among each other or to other non-target members?

[xmeng]

Hi,

Thank you for your kind reply.

There are NO "IdealGroupGenes.tsv" and "SecondChoiceGroupGenes.tsv" files in primer_group_out3. Only a "UndesiredGroupGenes.tsv" file is there. It looks like that these three members in the community are too distinct that they don’t share similar genes.

Again, thanks for your help.

[hghezzi]

Did you try designing microbe-specific primers for those 3 members? You could pool them to create a primer mix, which would essentially act as a group-specific primer set.

We have done something similar in the manuscript to detect the bacterial family Muribaculaceae and it worked well!