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# denovotranscript
  • a
    Can I get help from someone on the core team (@mashehu) to activate Zenodo functionality for this pipeline? I'm not sure how it works - do you need to manully write who the contributors are, or does it automatically get that info from the GitHub commits? The repo uses some ideas and code (from a previous repo) from @Timothy Little, but there aren't any commits from him directly. So, is it possible to add him when generating the DOI?
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  • a
    @Timothy Little could I get an approval for this pull request (only a few lines changed): https://github.com/nf-core/denovotranscript/pull/11
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  • c
    Hi all! Thank you for making this pipeline! I’m reasonably new to transcript assembly (some toe dipping with Stringtie) and am keen to try out your creation! I’ve got a couple of questions: • I’m using Seqera Platform to launch the pipeline. I’m using Trinity as the assembler. The field shown below suggests that I only need to set the strand-specificity for rnaSPAdes but I thought this would need to be set for Trinity as well?
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  • c
    • I am using a test set of 4 samples only. My runs currently fail on 
    NFCORE_DENOVOTRANSCRIPT:DENOVOTRANSCRIPT:TRINITY (pooled_reads)
    with a timeout error (16h). Is it expected for this step to take that long? Also, this is currently on 4 samples only - I was hoping to do a lot more eventually. Since the samples are merged in the first step will the time requirement for more samples then become prohibitive? Any thoughts would be much appreciated!
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  • r
    Hi guys! Thank you very much for the pipeline :) I would like to ask @Avani Bhojwani a question. I am currently trying to run denovotranscript on my institute's cluster, and I am getting an error during the BUSCO - metaeuk step. I am attaching nextflow.log and busco_config.ini . I am not really sure how to fix this error (maybe I need to specify metaeuk path differently in the config file? 🤔). I wonder if you could have a quick look, maybe it would be easier for you to spot the problem.
    busco_config.ini.nextflow.log
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  • s
    Faridi Lab has joined this channel by invitation from nf-core.
  • i
    Hi Everyone 🙂 I'm running this pipeline on a slurm cluster with Singularity and am trying to get set up to use --grid_exec. • If I configure the param for extra trinity args as: 
    "extra_trinity_args": "--SS_lib_type RF --grid_exec \"sbatch --qos shortq --time 0:29:59 --mem 1GB --cpus-per-task 1\"",
    I get the error: 
    bash: line 1: sbatch: command not found
    . • Reading further, I've seen that it's recommended to use HPCGridRunner to distribute the commands in 
    recursive_trinity.cmds
    as jobs. I've tried this approach, but get the error (with paths shortened):
    Copy code
    *** Dispatching parallel commands to the compute farm:
Friday, January 17, 2025: 16:11:21	CMD: .../hpc_cmds_GridRunner.pl -G .../hpc_conf/SLURM.Monash.conf -c recursive_trinity.cmds
SERVER: m3v117, PID: 24318
FARMIT failed to accept job: sbatch --qos shortq --time 0:29:59 --mem 1GB --cpus-per-task 1 -e .../pooled_reads_trinity/farmit.J24318.m3v117.24318.1737090681/cmds/J24318.S0.sh.stderr -o .../pooled_reads_trinity/farmit.J24318.m3v117.24318.1737090681/cmds/J24318.S0.sh.stdout .../pooled_reads_trinity/farmit.J24318.m3v117.24318.1737090681/cmds/J24318.S0.sh 2>&1 
 (ret -1)
FARMIT failed to accept job.  Will try again shortly.
    • I thought that the above attempts fail because Trinity is being run within a Singularity container and so 
    sbatch
    isn't available, but the error message mentioning 
    FARMIT
    comes from HpcGridRunner - not sure how it'd be reached if the execution environment was inside the container. • I've tried running HpcGridRunner separately on 
    recursive_trinity.cmds
    and it worked to distribute jobs - but they all failed because they're configured for the path to Trinity inside the container (duh 🤦‍♂️). • I came across these threads with Brian's responses seemingly about this issue, but it's not clear to me if his changes have made it to the current version of Trinity v2.15.2. https://github.com/trinityrnaseq/trinityrnaseq/issues/952 and https://groups.google.com/g/trinityrnaseq-users/c/-DnnDYKe0xo/m/Lp6ZUF-QAwAJ Happy to give up on 
    grid_exec
    for now if it's just too much trouble, but would be curious to hear if anybody else has had success (or can see something I've missed!). Thank you! 😄
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  • i
    Hi all, wondering if there's an option to run multiple samples without them being concatenated? Or do we need to run the pipeline multiple times, one per sample, if we want to keep the assemblies separate?
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  • e
    Hello everyone! I would like to support a way to improve this pipeline. First of all, I would like to thank all contributors as well as the people who originally created this pipeline, because it provides a great starting point for de-novo transcriptome assembly. Currently, this pipeline arrives at an assembled transcriptome that doesn't have particularly helpful annotations, since Trinity gives its own IDs to transcripts that don't look like the known ones. The missing step is to annotate these transcripts (e.g. with BRAKER3 or Augustus), make predictions for Gene Ontology associations, find protein sequences, protein domains etc. Such a single pipeline does not exist in nf-core, but it does exist in Snakemake (eg., https://onlinelibrary.wiley.com/doi/10.1111/1755-0998.13593 and https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-023-05254-8). It is not necessarily novel in the sense that no one else has thought of it, but the fact remains that it does not exist in nf-core. We could possibly add other best-practices to the analysis, such as PCR artifact deduplication, or even giving reference organisms and finding homologs to make a very comprehensive pipeline. There is also this paper on the steps of de-novo transcriptome assembly. https://academic.oup.com/bib/article/23/2/bbab563/6514404 It would be my pleasure to contribute to this task and improve this current implementation. I believe it would be helpful for a lot of scientists especially now since there is a mass sequencing of new genomes.
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  • e
    I'm also up for assisting with these changes. Let us know if that's ok, so we can start developing some new nf-core modules and adding them to this pipeline 😉 We can always make a call to direct and prioritise efforts
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  • a
    Thank you for your suggestions @Eleftherios Bochalis and @Evangelos Karatzas. I am really happy to hear that there are people willing to contribute to the development of the pipeline. I agree that there is plenty of room for improvement to make it more useful. Because I started working on this pipeline as a part of my PhD project, I will just need to check with my supervisor about some things. I will aim get back to you about this in a few days
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  • e
    And let's prioritise which of the suggested changes should be carried out first 😉
  • a
    Looks amazing! In terms of priorities, I'm thinking TransDecoder, InterProScan, Diamond, and Trinotate. What do you think?
  • e
    So, mainly focus on the missing subworkflow, which is the annotation part, rather than start the updates from start to end. Could do that. I made a list of the modules that we will need to nf-core, based on Eleftherios' suggestions:
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    jellyfish (C++), bioconda: <https://bioconda.github.io/recipes/kmer-jellyfish/README.html#package-package%20&#x27;kmer-jellyfish&#x27>; 
transrate (Ruby), bioconda:
<https://bioconda.github.io/recipes/transrate/README.html#package-package%20&#x27;transrate&#x27>; 
rnaquast (python), bioconda:
<https://bioconda.github.io/recipes/rnaquast/README.html#package-package%20&#x27;rnaquast&#x27>; 
trnascan-se (C/perl): bioconda:
<https://bioconda.github.io/recipes/trnascan-se/README.html#package-package%20&#x27;trnascan-se&#x27>; 
trinotate (perl/JS): bioconda:
<https://bioconda.github.io/recipes/trinotate/README.html#package-package%20&#x27;trinotate&#x27>;
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  • e
    I'll start with trinotate then, and will play a bit with the pipeline to figure out which channel to feed to transdecoder
  • e
    Probably the Trinity output?
  • a
    the final assembly is produced by the EVIGENE_TR2AACDS module. In the denovotranscript.nf workflow, I have created ch_transcripts which contains the fasta file with rna sequences - this is probably what you want to use for transdecoder edit: I just realised I will need to look into how Evidential Gene deals with RNA that's not mRNA. I've mainly been focusing on mRNA, but I noticed some of the annotation steps apply to other types too
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  • e
    `jellyfish/count`added to nf-core: https://github.com/nf-core/modules/tree/master/modules/nf-core/jellyfish/count Will also upload `jellyfish/dump`soon,to get the human readable output
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  • e
    I tried to standardise 
    rnaquast
     as an nf-core module, and here is the PR: https://github.com/nf-core/modules/pull/8432 I know it can have more input files, but these are the 3 (transcripts, reference, gtf) that the local module was also using, so I would like to see in a first iteration, if its container/environment is sufficient to produce similar results to yours (contains the same libs with no errors). If possible, have a look at the PR and let me know
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  • e
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    Template was merged. Will continue on a new branch with some linting fixes up next
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  • e
    Hi @Avani Bhojwani, can you make me a co-moderator for the pipeline? Need to rename from master to main, according to this video:

    https://www.youtube.com/watch?v=WhpPh8PvZp0&amp;t=7s&amp;ab_channel=nf-core▾

    (Already done this for my proteinfamilies pipeline)
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  • e
    The default branch for denovotranscript has been renamed from 
    master
    to 
    main
    . If you have a local clone, you can update it by running the following commands:
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    git branch -m master main
git fetch origin
git branch -u origin/main main
git remote set-head origin -a
  • e
    lint warnings resolve + 
    toolCitationText
    and 
    toolBibliographyText
    for reporting PR: https://github.com/nf-core/denovotranscript/pull/44
  • s
    Hello, Thank you for creating the pipeline. As a newcomer to nf-core, I am attempting to run this pipeline for de novo assembly of Oak transcripts. I was able to successfully execute the pipeline up to the Trinity and rnaSPAdes steps, but it consistently fails at the 
    tr2aacds.pl
    step. Specifically, the issue seems to be with the BLAST commands. I have attached the log file and a screenshot of the directory where the temporary BLAST outputs are being stored. Could you please let me know what might be causing this error?
  • s
    Hi All, Thanks so much for this pipeline, I'm looking forward to using it! I downloaded fastq files from SRA, which aren't gzipped. Is it possible to run the pipeline with un-gzipped files? I got an error that the validation of pipeline parameters failed, because they didn't end in .gz; I'm looking through the documentation and nothing explicitly says they need to be gzipped, and my understanding is that other nf-core pipelines can accept non-gzipped files. Is there some kind of flag I can pass so that I don't need to additional gzip all the files when they're already uncompressed? Thanks!
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  • s
    This feels like a silly question -- does the pipeline assume that all samples in the samplesheet are from the same individual/species? For context, for my project I'm assembling a bunch of transcriptomes from multiple species. Since we're only interested in a few genes, we're only using one sample per species, so without thinking I dumped them in the same samplesheet. Do I need to have a separate samplesheet for each species' sample?
  • s
    Hi <!channel> sorry for the ping, but I really do need an answer to the above question. I assumed that I'm correct and that each run of the pipeline assumes all samples are from the same species, because of the step where it pools all the runs. I started running them separately while waiting for an answer, but I have enough species (57) that I am running into the file number limit on my scratch because of how many intermediate files are generated (somehow even 10 runs at a time generates 1M files??). If I'm wrong and could be running this as one big pipeline, that would be great to know. Thanks, Serena
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  • g
    Hi, I am getting this error message. The pipeline seemed to be working fine earlier:
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    curl: (60) Peer's Certificate has expired.
More details here: <http://curl.haxx.se/docs/sslcerts.html>

curl performs SSL certificate verification by default, using a "bundle"
 of Certificate Authority (CA) public keys (CA certs). If the default
 bundle file isn't adequate, you can specify an alternate file
 using the --cacert option.
If this HTTPS server uses a certificate signed by a CA represented in
 the bundle, the certificate verification probably failed due to a
 problem with the certificate (it might be expired, or the name might
 not match the domain name in the URL).
If you'd like to turn off curl's verification of the certificate, use
 the -k (or --insecure) option.

 N E X T F L O W   ~  version 25.10.2

ERROR ~ Unable to parse config file: '/home/gthemudo/.nextflow/assets/nf-core/denovotranscript/nextflow.config'

  Cannot read config file include: <https://raw.githubusercontent.com/nf-core/configs/master/nfcore_custom.config>
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  • l
    Hi I am trying to run command on local Ubuntu server using Singularity and hitting a persistent error before any processes launch. 
    ~/software/nextflow run "$PIPELINE" \
    --work-dir "$WORKDIR" \
    --input "$BASE/src/CP_primary_files.csv" \
    --outdir "$BASE/results/plurivora_denovo" \
    --skip_fastp \
    --skip_fastqc \
    --assemblers trinity_no_norm \
    --extra_trinity_args "--SS_lib_type RF --jaccard_clip" \
    --busco_lineage 'stramenopiles_odb10' \
    -profile singularity \
    -resume \
    2>&1 | tee "$LOG"
    Every run completes with 
    succeededCount=0; failedCount=0
     — no processes ever start. When I had SORTMERNA, it always shows 
    work-dir=null
    .I downloaded the earlier version of NextFlow (25.04.4) with pipeline version (v1.2.1) to try and manage this and have tried a variety of tiny fixes. From this script, I am getting the issue of ERROR ~ Cannot invoke “java.nio.file.Path.getFileSystem()” because “path” is null -- Check script ‘/home/c838579834/software/nfcore_denovotranscript/subworkflows/nf-core/utils_nfcore_pipeline/main.nf’ at line: 82 or see ‘.nextflow.log’ file for more details [- ] NFCORE_DENOVOTRANSCRIPTDENOVOTRANSCRIPTCAT_FASTQ - [- ] NFCORE_DENOVOTRANSCRIPTDENOVOTRANSCRIPTTRINITY_NO_NORM - [- ] NFCORE_DENOVOTRANSCRIPTDENOVOTRANSCRIPTCAT_CAT - [- ] NFCORE_DENOVOTRANSCRIPTDENOVOTRANSCRIPTEVIGENE_TR2AACDS - [- ] NFCORE_DENOVOTRANSCRIPTDENOVOTRANSCRIPTTX2GENE - [- ] NFCORE_DENOVOTRANSCRIPTDENOVOTRANSCRIPTBUSCO_BUSCO - [- ] NFCORE_DENOVOTRANSCRIPTDENOVOTRANSCRIPTRNAQUAST - [- ] NFCORE_DENOVOTRANSCRIPTDENOVOTRANSCRIPTTRANSRATE - [- ] NFCORE_DENOVOTRANSCRIPTDENOVOTRANSCRIPTSALMON_INDEX - [- ] NFCORE_DENOVOTRANSCRIPTDENOVOTRANSCRIPTSALMON_QUANT - [- ] NFCORE_DENOVOTRANSCRIPTDENOVOTRANSCRIPTMULTIQC - Pulling Singularity image depot.galaxyproject.org/singularity/ubuntu:20.04 [cache /home/c838579834/singularity_cache/depot.galaxyproject.org-singularity-ubuntu-20.04.img] ERROR ~ Cannot invoke “java.nio.file.Path.getFileSystem()” because “path” is null Thoughts? Thanks.
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