Enrich backbone tree with 16S

[Ge94]

Hello,

I am trying to reproduce an approach similar to what GreenGenes2 performed through uDance. I believe I am missing a step, or I haven't understood the paper correctly, could you kindly guide me through this?

I generated a bacterial phylogenomic tree of MAGs and complete isolates with phylophlan, to which I would like to add new 16S sequences. I therefore generated an msa of 16S sequences, and 400 more msas of the genomes based on the 400 phylophlan markers. I then launched uDance under the "backbone: tree" configuration (not "de-novo") and I am currently stuck at this error:

Traceback (most recent call last):
  File "/hps/nobackup/rdf/metagenomics/service-team/users/germanab/uDance/run_udance.py", line 10, in <module>
    options.func(options)
  File "/hps/nobackup/rdf/metagenomics/service-team/users/germanab/uDance/uDance/decompose.py", line 148, in decompose
    aggregate_placements(index_to_node_map, jp['placements'])
  File "/hps/nobackup/rdf/metagenomics/service-team/users/germanab/uDance/uDance/decompose.py", line 27, in aggregate_placements
    index_to_node_map[index].placements += [seqname]
KeyError: -1

My understanding is that this comes from apples2, as it wasn't able to place the first of my 16S sequences:

Taxon AB025012.1 cannot be placed. At least three non-infinity distances should be observed to place a taxon. Consequently, this taxon is ignored (no output).

I have a couple concerns from this:

• apples2 stopped reading query sequences after the first (which failed), even if it's input query.fa contains 460. Shouldn't ignore a query sequence and proceed with the following ones?
• apples2 error about needing three points to compute a distance absolutely make sense, but how can I compute distances differently, if complete 16S sequences are aligned only to each other (e.g. they don't match any of the phylophlan markers of course)? Should I generate other msas with those sequences? Since the 16S msa contains both new 16S query sequences, and 16S sequences extracted from the genomes in the backbone tree, I thought that the alignment itself would have served for the computation of multiple distances.

Thank you in advance for your help. I'd be happy to clarify any of my doubts further.

Best
Germana

[balabanmetin]

Hello Germana, I believe you are experiencing this issue because you are using the whole genome version of uDance. In Greengenes2 paper, we use a special version of uDance that grows a tree using a single (16S) gene. That version is listed under releases: https://github.com/balabanmetin/uDance/tree/v1.1.0 https://github.com/balabanmetin/uDance/releases/tag/v1.1.0 Try to use this version and if you are stuck, let me know

…

On Thu, Aug 14, 2025, 8:43 AM Germana Baldi ***@***.***> wrote: *Ge94* created an issue (balabanmetin/uDance#15) <#15> Hello, I am trying to reproduce an approach similar to what GreenGenes2 performed through uDance. I believe I am missing a step, or I haven't understood the paper correctly, could you kindly guide me through this? I generated a bacterial phylogenomic tree of MAGs and complete isolates with phylophlan, to which I would like to add new 16S sequences. I therefore generated an msa of 16S sequences, and 400 more msas of the genomes based on the 400 phylophlan markers. I then launched uDance under the "backbone: tree" configuration (not "de-novo") and I am currently stuck at this error: Traceback (most recent call last): File "/hps/nobackup/rdf/metagenomics/service-team/users/germanab/uDance/run_udance.py", line 10, in <module> options.func(options) File "/hps/nobackup/rdf/metagenomics/service-team/users/germanab/uDance/uDance/decompose.py", line 148, in decompose aggregate_placements(index_to_node_map, jp['placements']) File "/hps/nobackup/rdf/metagenomics/service-team/users/germanab/uDance/uDance/decompose.py", line 27, in aggregate_placements index_to_node_map[index].placements += [seqname] KeyError: -1 My understanding is that this comes from apples2, as it wasn't able to place the first of my 16S sequences: Taxon AB025012.1 cannot be placed. At least three non-infinity distances should be observed to place a taxon. Consequently, this taxon is ignored (no output). I have a couple concerns from this: - apples2 stopped reading query sequences after the first (which failed), even if it's input query.fa contains 460. Shouldn't ignore a query sequence and proceed with the following ones? - apples2 error about needing three points to compute a distance absolutely make sense, but how can I compute distances differently, if complete 16S sequences are aligned only to each other? Should I generate other msas with those sequences? Thank you in advance for your help. Best Germana — Reply to this email directly, view it on GitHub <#15>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABRDOXK25OO7WPQC4Y2GGET3NSVAZAVCNFSM6AAAAACD5EIIUKVHI2DSMVQWIX3LMV43ASLTON2WKOZTGMZDENRYGM4DIMA> . You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

[Ge94]

Hi Metin,

Thank you for your very quick response. It took me a while to reinstall the tool, set up the new experiment and debug the results.

Unfortunately, I am indeed stuck at dedup.sh, more specifically at line 17

python $SCRIPTS_DIR/order_id.py --backbone ${temp_dir}/backbone_id.txt --all ${temp_dir}/all_id.txt --out-dir ${temp_dir}

Which fails with the following error:
Missing backbone sequences in alignment!
Missing: { [list of sequences ] }

[Ge94]

Sorry, I sent the message too early!

As I previously mentioned my backbone is composed of both MAGs and isolates, some of them do not contain any 16S sequences, and it's physically impossible to align the new query 16S sequences to all genome, as only few 16S sequences were extracted from the genomes in the backbone. Is there anything I am missing - for example, should I launch uDance only on sequences that actually have a 16S gene, or is there a workaround I missed?

Thank you very much in advance

Germana