I plan to add full classification capability to the pipeline. I've made an issue to describe this: https://github.com/nf-core/phyloplace/issues/18. Anyone with an interest, please comment.

Please see my little announcement in #CEA7TBJGJ regarding reference trees for archaeal and bacterial 16S sequences: https://nfcore.slack.com/archives/CEA7TBJGJ/p1719412715953679

YAML for ☝️ for #C04NLA7QHH9 params: Archaea:

queryseqfile: 'archaeal_sequences.fna'
refphylogeny: '<https://figshare.scilifelab.se/ndownloader/files/45818916>'
refseqfile: '<https://figshare.scilifelab.se/ndownloader/files/45818919>'
taxonomy: '<https://figshare.scilifelab.se/ndownloader/files/45828279>'
model: 'GTR+F+I+G4'

Bacteria:

queryseqfile: 'bacterial_sequences.fna'
refphylogeny: '<https://figshare.scilifelab.se/ndownloader/files/45818913> bac120.newick'
refseqfile: '<https://figshare.scilifelab.se/ndownloader/files/45818925> bac120.alnfna'
taxonomy: '<https://figshare.scilifelab.se/ndownloader/files/45828282> bac120.taxonomy.tsv'
model: 'GTR+F+I+G4'

Note that there's also a way of writing a

samplesheet.csv

with the above information:

sample,alignmethod,queryseqfile,refseqfile,refphylogeny,model,taxonomy
bacteria-r08rs214,mafft,bacterial_sequences.fna,<https://figshare.scilifelab.se/ndownloader/files/45818925,https://figshare.scilifelab.se/ndownloader/files/45818913,GTR+F+I+G4,https://figshare.scilifelab.se/ndownloader/files/45828282>
archaea-r08rs214,mafft,archaeal_sequences.fna,<https://figshare.scilifelab.se/ndownloader/files/45818919,https://figshare.scilifelab.se/ndownloader/files/45818916,GTR+F+I+G4,https://figshare.scilifelab.se/ndownloader/files/45828279>

Use with

--input

.

I’m new to the field of phylogenetics/genomics but my lab has tasked me with assembling genomes of bioprospected organisms and performing a phylogenetic analysis of them. Is nf-core/phyloplace a suitable pipeline to use for whole genome assemblies? If not, is there an alternative way to do phylogenetics when you have draft assemblies that’s more robust than 16/18s sequencing that anyone would recommend?

Phyloplace 2.0.0 was just released! The main new feature, which motivates the jump from 1.0.0 directly to 2.0.0, is that the pipeline can now be used to first search a fasta file with hmm profiles, then place significant hits in a phylogeny that corresponds to the hmm it hit best. The old mode of the pipeline -- "phyloplace" -- is kept, but for sheet input now uses the

--phyloplace_input

param, which retires the

--input

param. The new mode -- "phylosearch" -- uses the

--phylosearch_input

param. The nf-core homepage is not yet updated, but when it is: Check out the documentation there. My thanks to my reviewers: @Daniel Straub, @Maxime Garcia and @mashehu and every other nf-core member I pestered with questions!

Hi everyone, I was asking to Daniel in the github issues some questions on how to approach when you have AA reference sequences and want to explore metatranscriptomic (NT) reads: https://github.com/nf-core/phyloplace/issues/18#issuecomment-2657251266 From what you were saying @Daniel Lundin, you would use the

FeatureCounts

output from

metatdenovo

as a quantification then, after obtaining the ORFs and placing them. That makes quite a lot of sense. Since I was coming from the approaches made by

gappa

and

EPA

that focus mostly on placing reads I did not think on this approach. The ORFs however could be included directly in the alignment and tree avoiding EPA no? Or this would make everything slower. Thanks for your time!

Yes, using #C02FC6VFQG1 to reconstruct ORFs and quantify them (please check the output in the

results/summary_tables

, particularly

*.counts.tsv.gz

as this is nicely formatted) is the approach I'd advocate. When it comes to making the trees there's more to think about. You can certainly estimate a de novo phylogeny from the ORF sequences. Since your samples are unlikely to contain a good representation of the sequence space for each protein, however, placing in a reference tree (that was estimated from a balanced selection of sequences) is safer. It will also be faster since in phylogenetic placement, each sequence is placed individually. Resource requirements (cpu) will hence scale linearly with the number of sequences (memory is basically flat), which is not the case with full phylogenetics. The only drawback I see is that with placement you will not resolve the individual sequences' branching when they happen to branch off the reference tree at the same place. In other words, quite similar/closely related sequences will show up as a "multitomy", a brush in the resulting tree.

Ok thanks!

Keep the questions coming! 🙂

Hello, I've been creating a phylogenetic tree for the bacteria within my dataset using this code within the ampliseq pipeline: ""pplace_tree": "/cfs/klemming/projects/snic/naiss2023-22-1217/ampliseq_analysis/sbdi-gtdb-sativa.r09rs220.bac120.sprepr.brlenopt.newick", "pplace_aln": "/cfs/klemming/projects/snic/naiss2023-22-1217/ampliseq_analysis/sbdi-gtdb-sativa.r09rs220.bac120.sprepr.alnfna", "pplace_taxonomy": "/cfs/klemming/projects/snic/naiss2023-22-1217/ampliseq_analysis/sbdi-gtdb-sativa.r09rs220.bac120.sprep.taxonomy.tsv", "pplace_model": "GTR+F+I+G4", "pplace_name": "bac120.r09rs220"" However, now I saw that the tree is unrooted. How do I root the tree? Is there a function within the pipeline to do it? Thanks and kind regards!

Hello, I've been trying to create a phylogenetic tree using the ampliseq pipeline. When I use the following code, it works: "pplace_tree": "/cfs/klemming/projects/snic/naiss2023-22-1217/ampliseq_analysis/sbdi-gtdb-sativa.r09rs220.bac120.sprepr.brlenopt.newick", "pplace_aln":"/cfs/klemming/projects/snic/naiss2023-22-1217/ampliseq_analysis/sbdi-gtdb-sativa.r09rs220.bac120.sprepr.alnfna", "pplace_taxonomy":"/cfs/klemming/projects/snic/naiss2023-22-1217/ampliseq_analysis/sbdi-gtdb-sativa.r09rs220.bac120.sprep.taxonomy.tsv", "pplace_model": "GTR+F+I+G4", "pplace_name": "bac120.r09rs220" But when I want to use the newest version of these files, the "r10rs226" version, I get the following error message: ERROR ~ Error executing process > 'NFCORE_AMPLISEQAMPLISEQFASTA_NEWICK_EPANG_GAPPA:GAPPA_ASSIGN (bac120.r10rs226)' Caused by: Process

NFCORE_AMPLISEQ:AMPLISEQ:FASTA_NEWICK_EPANG_GAPPA:GAPPA_ASSIGN (bac120.r10rs226)

terminated for an unknown reason -- Likely it has been terminated by the external system Command executed: gappa \ examine assign \ --per-query-results --krona --sativa \ --threads 6 \ --jplace-path bac120.r10rs226.epa_result.jplace.gz \ --taxon-file sbdi-gtdb-sativa.r10rs226.bac120.sprep.taxonomy.tsv \ --file-prefix bac120.r10rs226.taxonomy. cat <<-END_VERSIONS > versions.yml "NFCORE_AMPLISEQAMPLISEQFASTA_NEWICK_EPANG_GAPPA:GAPPA_ASSIGN": gappa: $(echo $(gappa --version 2>&1 | sed 's/v//' )) END_VERSIONS Command exit status: - Command output: ...' .... ... ... ... ... .... .|'|. | || '' .|| ||' || ||' || '' .|| .|' || |'' .|' || || | || | .|' || .|'|. || '.... '|..'|'. ||...' ||...' '|..'|. '||' ||:. '....' || || '''' '''' v0.8.0 (c) 2017-2022 by Lucas Czech and Pierre Barbera Invocation: gappa examine assign --per-query-results --krona --sativa --threads 6 --jplace-path bac120.r10rs226.epa_result.jplace.gz --taxon-file sbdi-gtdb-sativa.r10rs226.bac120.sprep.taxonomy.tsv --file-prefix bac120.r10rs226.taxonomy. Command: gappa examine assign Input: --jplace-path bac120.r10rs226.epa_result.jplace.gz --taxon-file sbdi-gtdb-sativa.r10rs226.bac120.sprep.taxonomy.tsv --root-outgroup --taxonomy --ranks-string superkingdom|phylum|class|order|family|genus|species Settings: --sub-taxopath --max-level 0 --distribution-ratio -1 --consensus-thresh 1 --resolve-missing-paths false --distant-label false Output: --out-dir . --file-prefix bac120.r10rs226.taxonomy. --file-suffix --cami false --sample-id --krona true --sativa true --per-query-results true --best-hit false Global Options: --allow-file-overwriting false --verbose false --threads 6 --log-file Run the following command to get the references that need to be cited:

gappa tools citation Czech2020-genesis-and-gappa

Started 2025-09-21 143340 Found 1 jplace file Running the assignment Command error: INFO: Environment variable SINGULARITYENV_TMPDIR is set, but APPTAINERENV_TMPDIR is preferred INFO: Environment variable SINGULARITYENV_NXF_TASK_WORKDIR is set, but APPTAINERENV_NXF_TASK_WORKDIR is preferred INFO: Environment variable SINGULARITYENV_NXF_DEBUG is set, but APPTAINERENV_NXF_DEBUG is preferred INFO: fuse2fs not found, will not be able to mount EXT3 filesystems INFO: gocryptfs not found, will not be able to use gocryptfs Work dir: /cfs/klemming/projects/supr/naiss2025-22-1066/ampliseq/work/9f/08e866f71ac92b3d080d803f8261d7 Container: /cfs/klemming/projects/supr/naiss2025-22-1066/ampliseq/work/singularity/depot.galaxyproject.org-singularity-gappa-0.8.0--h9a82719_0.img Tip: you can replicate the issue by changing to the process work dir and entering the command

bash .command.run

-- Check '.nextflow.log' file for details ERROR ~ Pipeline failed. Please refer to troubleshooting docs: https://nf-co.re/docs/usage/troubleshooting -- Check '.nextflow.log' file for details ERROR ~ Failed to invoke

workflow.onComplete

event handler -- Check script '/cfs/klemming/home/n/nicolai2/.nextflow/assets/nf-core/ampliseq/subworkflows/local/utils_nfcore_ampliseq_pipeline/main.nf' at line: 123 or see '.nextflow.log' file for more details Can you help me with that?

Hej hej, I wanted to ask whether the update of the r226 is already available?

I'm afraid not. The first run finished, but still had some problems so I'm running it again. Good that you remind me, so I remember to post the info here (or, rather, #CEA7TBJGJ).

Hello there

I have an error with EPA and some doubts regarding how to handle it. The error:

Command error:
  INFO Selected: Output dir: ./
  INFO Selected: Query file: 022_180-2000_DCM.query.hmmalign.masked.afa.gz
  INFO Selected: Tree file: tree.nw
  INFO Selected: Reference MSA: 022_180-2000_DCM.ref.hmmalign.masked.afa.gz
  INFO Selected: Automatic switching of use of per rate scalers
  INFO Selected: Preserving the root of the input tree
  INFO Selected: Specified model: LG+R8
  INFO Selected: Using threads: 12
  INFO     ______ ____   ___           _   __ ______
          / ____// __ \ /   |         / | / // ____/
         / __/  / /_/ // /| | ______ /  |/ // / __  
        / /___ / ____// ___ |/_____// /|  // /_/ /  
       /_____//_/    /_/  |_|      /_/ |_/ \____/ (v0.3.8)
  INFO Using model parameters:
  INFO    Rate heterogeneity: FREE (8 cats, mean),  weights&rates: (0.125,0.0652803) (0.125,0.208627) (0.125,0.376074) (0.125,0.577429) (0.125,0.830101) (0.125,1.1699) (0.125,1.69315) (0.125,3.07944) 
          Base frequencies (model): 0.079066 0.055941 0.041977 0.053052 0.012937 0.040767 0.071586 0.057337 0.022355 0.062157 0.099081 0.0646 0.022951 0.042302 0.04404 0.061197 0.053287 0.012066 0.034155 0.069147 
          Substitution rates (model): 0.425093 0.276818 0.395144 2.48908 0.969894 1.03855 2.06604 0.358858 0.14983 0.395337 0.536518 1.12403 0.253701 1.17765 4.72718 2.1395 0.180717 0.218959 2.54787 0.751878 0.123954 0.534551 2.80791 0.36397 0.390192 2.4266 0.126991 0.301848 6.32607 0.484133 0.052722 0.332533 0.858151 0.578987 0.593607 0.31444 0.170887 5.07615 0.528768 1.69575 0.541712 1.43765 4.50924 0.191503 0.068427 2.14508 0.371004 0.089525 0.161787 4.00836 2.00068 0.045376 0.612025 0.083688 0.062556 0.523386 5.24387 0.844926 0.927114 0.01069 0.015076 0.282959 0.025548 0.017416 0.394456 1.24028 0.42586 0.02989 0.135107 0.037967 0.084808 0.003499 0.569265 0.640543 0.320627 0.594007 0.013266 0.89368 1.10525 0.075382 2.78448 1.14348 0.670128 1.16553 1.95929 4.12859 0.267959 4.81351 0.072854 0.582457 3.23429 1.67257 0.035855 0.624294 1.22383 1.08014 0.236199 0.257336 0.210332 0.348847 0.423881 0.044265 0.069673 1.80718 0.173735 0.018811 0.419409 0.611973 0.604545 0.077852 0.120037 0.245034 0.311484 0.008705 0.044261 0.296636 0.139538 0.089586 0.196961 1.73999 0.129836 0.268491 0.054679 0.076701 0.108882 0.366317 0.697264 0.442472 0.682139 0.508851 0.990012 0.584262 0.597054 5.30683 0.119013 4.14507 0.159069 4.27361 1.11273 0.078281 0.064105 1.03374 0.11166 0.232523 10.6491 0.1375 6.31236 2.59269 0.24906 0.182287 0.302936 0.619632 0.299648 1.70274 0.656604 0.023918 0.390322 0.748683 1.13686 0.049906 0.131932 0.185202 1.79885 0.099849 0.34696 2.02037 0.696175 0.481306 1.89872 0.094464 0.361819 0.165001 2.45712 7.8039 0.654683 1.33813 0.571468 0.095131 0.089613 0.296501 6.47228 0.248862 0.400547 0.098369 0.140825 0.245841 2.18816 3.15182 0.18951 0.249313
  INFO Output file: ./epa_result.jplace
  terminate called after throwing an instance of 'std::runtime_error'
    what():  Sequence with header '022_180-2000_DCM_ERR1712074.25146093_1;weight=1;' does not appear to have any non-gap sites!
  /scratch/data3/aauladell/project_big/phylotranscriptomics/work/aa/ba4d9e9061f842ee0c944df9a618b1/.command.sh: line 9:    41 Aborted                 (core dumped) epa-ng --model LG+R8 --threads 12 --query 022_180-2000_DCM.query.hmmalign.masked.afa.gz --ref-msa 022_180-2000_DCM.ref.hmmalign.masked.afa.gz --tree tree.nw

Work dir:
  /scratch/data3/aauladell/project_big/phylotranscriptomics/work/53/85520b58953e69bd4e91f9d0e0f79a

some of the reads consist indeed of only gaps, perhaps with only one nucleotide. But reading the wiki, it would seem that EPA should be able to throw them out:

By default,

EPA-ng

enables premasking, which works similarily to the same option in pplacer: If a site of the alignment is all-gaps in either the reference OR query alignment, throw it out. Further, for each query sequence, ignore the leading, and trailing gap columns (this is where we differ from pplacer, as they ignore ALL query gap columns).

from: https://github.com/pierrebarbera/epa-ng

any idea on why this is happening? Thanks a lot

Hej hej, I wanted to ask whether the update of the r226 is already available or if you know when you predict it to be available? Kind regards!

I think I will upload it this week. I haven't had access to the machine I'm running the pipeline in for a few days but soon I will

Morning. Finally I needed to include the

--no-pre-mask

flag since it happened for more than 10% of the samples was too much. The options are 1) to include the HMM cleaning as you said and 2) to add the flag, which works because I tested it with one sample. Given that these are a minority of reads I think its a good workaround, and sometimes the users won't have an specific HMM to use I think.This opened though a question when I tried to run the workflow adding by myself the tag. I thought that I should add it in the

nextflow.config

following this schema:

process {
    withName: 'EPANG_PLACE' {
        ext.args = ['--no-pre-mask']
    }
}

But if I add this, the software is unable to find the files, throwing this error.

ERROR ~ Argument of file function cannot be null

I am guessing that the call

ext.args

does not include the value upfront but substitutes partially the input that you give manually? This is my call:

nextflow run nf-core/phyloplace     --input data/placement_phyloplace/SIT/SIT_undone_samsheet.csv     -profile singularity --outdir data/placement_phyloplace/SIT

Thanks! PD: it is possible that the error comes from me using a previous version of nextflow (v23) or from the pipeline, I will be testing this, but if your intuition tells you that this is the problem just say that and sorry beforehand!)

I think it will be necessary to filter out in some way the output of hmmalign. Many of the results are mostly gaps and with the

--no-pre-mask

option it slows the overall performance. If I check the number of seqs in the output of hmmalign I can have something like this:

file  format  type     num_seqs    sum_len  min_len  avg_len  max_len
-     FASTA   Protein   250,212  7,200,646        1     28.8      610

(take into account that the length is with gaps removed) If I filter out any sequence below 15 aa I get something like this:

file  format  type     num_seqs    sum_len  min_len  avg_len  max_len
-     FASTA   Protein    66,256  6,846,372       15    103.3      610

It is quite a reduction. Could it be a step of the pipeline? Or else we should understand what is happening with hmmalign? Let me know what you think, thanks!

Hej hej, I have been trying to run phyloplace within ampliseq (2.16.1) and it terminates giving the following comments: -[nf-core/ampliseq] Pipeline completed with errors- ERROR ~ Error executing process > 'NFCORE_AMPLISEQAMPLISEQFASTA_NEWICK_EPANG_GAPPA:EPANG_PLACE (bac120.r10rs226)' Caused by: Process

NFCORE_AMPLISEQ:AMPLISEQ:FASTA_NEWICK_EPANG_GAPPA:EPANG_PLACE (bac120.r10rs226)

terminated for an unknown reason -- Likely it has been terminated by the external system Command executed: epa-ng \ --model GTR+F+I+G4 \ --threads 10 \ --query bac120.r10rs226.query.hmmalign.masked.afa.gz \ --ref-msa bac120.r10rs226.ref.hmmalign.masked.afa.gz \ --tree sbdi-gtdb-sativa.r10rs226-2.bac120.sprepr.brlenopt.newick \ \ if [ -e epa_result.jplace ]; then gzip epa_result.jplace cp epa_result.jplace.gz bac120.r10rs226.epa_result.jplace.gz fi [ -e epa_info.log ] && cp epa_info.log bac120.r10rs226.epa_info.log cat <<-END_VERSIONS > versions.yml "NFCORE_AMPLISEQAMPLISEQFASTA_NEWICK_EPANG_GAPPA:EPANG_PLACE": epang: $(echo $(epa-ng --version 2>&1) | sed 's/^EPA-ng v//') END_VERSIONS Command exit status: - Command output: INFO Selected: Output dir: ./ INFO Selected: Query file: bac120.r10rs226.query.hmmalign.masked.afa.gz INFO Selected: Tree file: sbdi-gtdb-sativa.r10rs226-2.bac120.sprepr.brlenopt.newick INFO Selected: Reference MSA: bac120.r10rs226.ref.hmmalign.masked.afa.gz INFO Selected: Automatic switching of use of per rate scalers INFO Selected: Preserving the root of the input tree INFO Selected: Specified model: GTR+F+I+G4 INFO Selected: Using threads: 10 INFO ______ __ _ _ __ ______ / ____// __ \ / | / | / // ____/ / __/ / /_/ // /| | ______ / |/ // / __ / /___ / ____// _ |/_____// /| // /_/ / /_____//_/ /_/ |_| /_/ |_/ \____/ (v0.3.8) INFO Using model parameters: INFO Rate heterogeneity: GAMMA (4 cats, mean), alpha: 1 (ML), weights&rates: (0.25,0.136954) (0.25,0.476752) (0.25,1) (0.25,2.38629) P-inv (ML): 0 Base frequencies (empirical): 0.254302 0.200097 0.338562 0.207039 Substitution rates (ML): 0.5 0.5 0.5 0.5 0.5 1 INFO Output file: ./epa_result.jplace Command error: INFO: Environment variable SINGULARITYENV_TMPDIR is set, but APPTAINERENV_TMPDIR is preferred INFO: Environment variable SINGULARITYENV_NXF_TASK_WORKDIR is set, but APPTAINERENV_NXF_TASK_WORKDIR is preferred INFO: Environment variable SINGULARITYENV_NXF_DEBUG is set, but APPTAINERENV_NXF_DEBUG is preferred INFO: fuse2fs not found, will not be able to mount EXT3 filesystems INFO: gocryptfs not found, will not be able to use gocryptfs Work dir: /cfs/klemming/projects/supr/naiss2025-22-1066/ampliseq/work/f8/5d7a34040c5551fd0a55eb9b7a8a89 Container: /cfs/klemming/projects/supr/naiss2025-22-1066/ampliseq/work/singularity/depot.galaxyproject.org-singularity-epa-ng-0.3.8--h9a82719_1.img Tip: when you have fixed the problem you can continue the execution adding the option

-resume

to the run command line -- Check '.nextflow.log' file for details ERROR ~ Pipeline failed. Please refer to troubleshooting docs: https://nf-co.re/docs/usage/troubleshooting -- Check '.nextflow.log' file for details My params-file looks like this: { "input": "manifest_BS.tsv", "skip_cutadapt": true, "outdir": "results_new", "pplace_tree": "/cfs/klemming/projects/snic/naiss2023-22-1217/ampliseq_analysis/SBDI_GTDB_v11/sbdi-gtdb-sativa.r10rs226-2.bac120.sprepr.brlenopt.newick", "pplace_aln": "/cfs/klemming/projects/snic/naiss2023-22-1217/ampliseq_analysis/SBDI_GTDB_v11/sbdi-gtdb-sativa.r10rs226-2.bac120.sprepr.alnfna", "pplace_taxonomy": "/cfs/klemming/projects/snic/naiss2023-22-1217/ampliseq_analysis/SBDI_GTDB_v11/sbdi-gtdb-sativa.r10rs226-2.bac120.sprepr.taxonomy.tsv", "pplace_model": "GTR+F+I+G4", "pplace_name": "bac120.r10rs226", "skip_qiime_downstream": true, "skip_barplot": true, "skip_abundance_tables": true, "skip_alpha_rarefaction": true, "skip_ancom": true, "picrust": false } Can you please help me with this? Thank you!

✅ phyloplace/test_full completed successfully! 🎉 Run Name wise_ardinghelli Duration 17m 16s Status ✅ Success Tasks Completed: 12 Jun 15, 2026 at 1:00 PM

✅ phyloplace/test_full completed successfully! 🎉 Run Name ecstatic_coulomb Duration 7m 51s Status ✅ Success Tasks Completed: 12 Jun 16, 2026 at 9:02 AM

<!channel>: New release: 2.0.1 out now. Template and software updates plus some docs improvements, changelog here: https://github.com/nf-core/phyloplace/blob/master/CHANGELOG.md

✅ phyloplace/test_full completed successfully! 🎉 Run Name gigantic_kalam Duration 3m 28s Status ✅ Success Tasks Completed: 12 Jul 24, 2026 at 3:59 PM

<!channel>: New release: 2.1.0 out now. Template update and improvement to MultiQC output, changelog here: github.com/nf-core/phyloplace/blob/…/CHANGELOG.md

<!channel> Friday release! 2.2.0 out with a focus on HMMER classification turning the pipeline into a better general classification tool. Thanks @piplus2, @Danilo Di Leo, @Niklas Schandry, @Joon Klaps & @Diego Alvarez Saravia for your reviews! CHANGELOG: github.com/nf-core/phyloplace/blob/…/CHANGELOG.md

<!channel>: Another release, now 2.3.0 with the primary goal of improving handling of HMMER-derived domains. Thanks to @Joon Klaps, @Sébastien Guizard, @Jonathan Manning, @piplus2 and @auguste.gardette for your reviews. CHANGELOG: github.com/nf-core/phyloplace/blob/…/CHANGELOG.md