#CTC73HT8C I have another question, I searched on internet but did not fully understand. How could I specify how much resource like cpu and memory should be used for a perticular nextflow nf-core pipeline run on uppmax ? only specify the max_cpu max_memory ? or should I create a config files for each job?

#CTC73HT8C @Tobias Neumann I am useing nf-core/slamseq pipeline. I have encounter an constant error when I specify the “--skip_trimming true ” parameter .  If I do not skip and use the trim option ,then there is no problem. the error message .

No such variable: Exception evaluating property 'reads' for nextflow.util.ArrayTuple, Reason: groovy.lang.MissingPropertyException: No such property: reads for class: sun.nio.fs.UnixPath
Possible solutions: class

 -- Check script '/crex/proj/sllstore2017018/private/project/Trans_mem/nextflow/assets/nf-core/slamseq/main.nf' at line: 351 or see '.nextflow.log' file for more details
-[nf-core/slamseq] Pipeline completed with errors-
WARN: Killing pending tasks (1)

And corresponding code in line 351 as following

/* 347 * STEP 1 - TrimGalore! 348 / 349 if (params.skip_trimming) { 350 rawFiles 351 .map{ it -> return tuple(it, file(it.reads)) } 352 .set{ trimmedFiles } 353 trimgaloreQC = Channel.empty()

Is this the place that I should ask for help???? Or is where should I report the issue? my first time to use slack, exploring how to use it. so if this message bothers you ,I am really sorry

Hm looks like it might be an error in the design input file

can you send this?

yes this is one of the places you could ask btw 🙂

@Tobias Neumann Hi, I have report the issue on Github. I tried to bypass the issue with parameter “--trim5 0” but apparently this is not a good idea at all. Glad to joint here 🤣 🤣 🤣 🤣 🤣 🤣

Yes it looks like it’s a problem in Nextflow

Which I think is related to an improperly formatted design file

@Tobias Neumann Happy to try after you guys fix it. BTW, this pipeline is really nice and the nextflow actually put an end to my nightmare of pipeline design. Slamdunk is a great solution for majority of 4sU labelling. I actually encountered a problem that until now I am still confused: the slamdunk only calls the T->C conversion on reads1 + A->G conversion on reads2 . BUT, there is no option that I could call A->G conversion on read1 and T->C conversion on read2. I am not very strong in bioinformatics so I am not very sure about my conclusion 🤣

now slamdunk calls T>C conversions on sense and A>G conversions on antisense reads, it does not related to mate-pair information

so can you send your input design.txt file so I can have a look at it?

one minute

@Tobias Neumann @hpatel I am getting this error when I am trying to run the pipeline. Any insights? I see something similar posted before but I do not see any answers. Thank you for your help!

Tip: when you have fixed the problem you can continue the execution adding the option -resume to the run command line

executor >  local (26)
[81/50e924] process > get_software_versions          [100%] 1 of 1 :heavy_check_mark:
[8f/d69887] process > checkDesign (design.tsv)       [100%] 1 of 1 :heavy_check_mark:
[c0/998697] process > trim (mESC-LNA_MALAT_1d-4SU... [100%] 8 of 8 :heavy_check_mark:
[ac/b951fe] process > map (mESC-LNA_MALAT_1d-4SU-... [ 63%] 5 of 8
[b9/c0596b] process > filter (mESC-CNTR_1d-4SU-rep3) [100%] 3 of 3
[be/ba3586] process > snp (mESC-LNA_MALAT_1d-4SU-... [100%] 2 of 2
[3b/f6cf6e] process > count (mESC-LNA_MALAT_1d-4S... [100%] 1 of 1
[-        ] process > collapse                       -
[43/add294] process > rates (mESC-LNA_MALAT_1d-4S... [100%] 1 of 1
[6a/b46521] process > utrrates (mESC-LNA_MALAT_1d... [100%] 1 of 1
[ee/834121] process > tcperreadpos (mESC-LNA_MALA... [100%] 1 of 1, failed: 1
[60/99fabb] process > tcperutrpos (mESC-LNA_MALAT... [100%] 1 of 1
[-        ] process > summary                        -
[-        ] process > deseq2                         -
[-        ] process > multiqc                        -
[87/2d46ae] process > output_documentation           [100%] 1 of 1 :heavy_check_mark:
Error executing process > 'tcperreadpos (mESC-LNA_MALAT_1d-4SU-rep1)'

Caused by:
  Process tcperreadpos (mESC-LNA_MALAT_1d-4SU-rep1) terminated with an error exit status (1)

Command executed:

  alleyoop tcperreadpos \
      -o tcperreadpos \
      -r genome.fa \
      -s .  \
      -mq 20 \
      -l 50 \
      -t 1 \
      mESC-LNA_MALAT_1d-4SU-rep1_trimmed.fq_slamdunk_mapped_filtered.bam

Command exit status:
  1

Command output:
  (empty)

Command error:
  Creating output directory: tcperreadpos
  Running alleyoop tcperreadpos for 1 files (1 threads)
  Traceback (most recent call last):
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/bin/alleyoop", line 10, in <module>
      sys.exit(run())
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/slamdunk/alleyoop.py", line 559, in run
      results = Parallel(n_jobs=n, verbose=verbose)(delayed(runTcPerReadPos)(tid, args.bam[tid], referenceFile, minMQ, args.maxLength, outputDirectory, snpDirectory, vcfFile) for tid in range(0, len(args.bam)))
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/parallel.py", line 1004, in call
      if self.dispatch_one_batch(iterator):
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/parallel.py", line 835, in dispatch_one_batch
      self._dispatch(tasks)
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/parallel.py", line 754, in _dispatch
      job = self._backend.apply_async(batch, callback=cb)
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/_parallel_backends.py", line 209, in apply_async
      result = ImmediateResult(func)
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/_parallel_backends.py", line 590, in init
      self.results = batch()
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/parallel.py", line 256, in call
      for func, args, kwargs in self.items]
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/parallel.py", line 256, in <listcomp>
      for func, args, kwargs in self.items]
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/slamdunk/alleyoop.py", line 232, in runTcPerReadPos
      stats.tcPerReadPos(referenceFile, bam, minMQ, maxReadLength, outputCSV, outputPDF, inputSNP, log)
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/slamdunk/dunks/stats.py", line 625, in tcPerReadPos
      tcCounts[mismatch.readPosition] += 1
  IndexError: list index out of range

hm what is your read length? is it maybe set too short?

Hello. Am I right to understand that duplicated reads were retained? Thanks!

Yepp unless you have sorted them out via UMI before, Slamdunk itself does not filter any duplicated reads.

Hi all, this might not be a slamseq specific error, but I’ll ask here just in case. I’m running using the docker profile, and the test profile also worked fine. I’m trying to run the following command:

nextflow run slamseq -profile docker -r 1.0.0 --input sampleList2.tsv --genome GRCm38 --bed genes.3utr.bed --max_cpus 6 --max_memory '20.GB'

but I keep getting the following error message:

Error executing process > 'tcperreadpos (20220111_A-o26962_1_20-xDR060_20_R1)'

Caused by:
  Process `tcperreadpos (20220111_A-o26962_1_20-xDR060_20_R1)` terminated with an error exit status (1)

Command executed:

  alleyoop tcperreadpos \
      -o tcperreadpos \
      -r genome.fa \
      -s .  \
      -mq 27 \
      -l 50 \
      -t 1 \
      20220111_A-o26962_1_20-xDR060_20_R1_trimmed.fq_slamdunk_mapped_filtered.bam

Command exit status:
  1

Command output:
  (empty)

Command error:
  WARNING: Your kernel does not support swap limit capabilities or the cgroup is not mounted. Memory limited without swap.
  Creating output directory: tcperreadpos
  Running alleyoop tcperreadpos for 1 files (1 threads)
  Traceback (most recent call last):
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/bin/alleyoop", line 10, in <module>
      sys.exit(run())
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/slamdunk/alleyoop.py", line 559, in run
      results = Parallel(n_jobs=n, verbose=verbose)(delayed(runTcPerReadPos)(tid, args.bam[tid], referenceFile, minMQ, args.maxLength, outputDirectory, snpDirectory, vcfFile) for tid in range(0, len(args.bam)))
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/parallel.py", line 1004, in __call__
      if self.dispatch_one_batch(iterator):
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/parallel.py", line 835, in dispatch_one_batch
      self._dispatch(tasks)
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/parallel.py", line 754, in _dispatch
      job = self._backend.apply_async(batch, callback=cb)
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/_parallel_backends.py", line 209, in apply_async
      result = ImmediateResult(func)
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/_parallel_backends.py", line 590, in __init__
      self.results = batch()
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/parallel.py", line 256, in __call__
      for func, args, kwargs in self.items]
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/joblib/parallel.py", line 256, in <listcomp>
      for func, args, kwargs in self.items]
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/slamdunk/alleyoop.py", line 232, in runTcPerReadPos
      stats.tcPerReadPos(referenceFile, bam, minMQ, maxReadLength, outputCSV, outputPDF, inputSNP, log)
    File "/opt/conda/envs/nf-core-slamseq-1.0.0/lib/python3.7/site-packages/slamdunk/dunks/stats.py", line 636, in tcPerReadPos
      totalReadCountRev[i] += 1
  IndexError: list index out of range

Work dir:
  /home/ubuntu/MOUNT3/davide/slamSeq/xDR060/nextflow/work/e5/2d496f2529b7b512a38780d9f0169d

Tip: when you have fixed the problem you can continue the execution adding the option `-resume` to the run command line

Any thoughts or insights?

Hi <!channel>, as you probably know already we have a coming hackathon code This edition will be focused on documentation 🗃️ And we need your help 🙏 If you feel like this pipeline is missing any documentation, can you please make a GitHub issue on its repo with the

documentation

label. We will sort out if it's dependent on the template or the pipeline itself and populate the hackathon project board accordingly. Thanks a lot for your help, On behalf of the hackathon organizing team nf core socks Maxime

@Tobias Neumann Hi Tobias, Can

slamdunk

accept a bam that is aligned by an external aligner such as

STAR

? I have a STAR-aligned bam as an input to

slamdunk filter

and below is error message. Thanks a lot!

File "/home/louella/miniconda3/envs/R-stable/lib/python3.9/site-packages/slamdunk/slamdunk.py", line 170, in runFilter
    filter.Filter(bam, outputBAM, getLogFile(outputLOG), bed, mq, minIdentity, maxNM, printOnly, verbose)
  File "/home/louella/miniconda3/envs/R-stable/lib/python3.9/site-packages/slamdunk/dunks/filter.py", line 270, in Filter
    mappedReads, unmappedReads, filteredReads, mqFiltered, idFiltered, nmFiltered, multimapper = multimapUTRRetainment (infile, outfile, bed, minIdentity, NM, log)
  File "/home/louella/miniconda3/envs/R-stable/lib/python3.9/site-packages/slamdunk/dunks/filter.py", line 107, in multimapUTRRetainment
    if(float(read.get_tag("XI")) < minIdentity):
  File "pysam/libcalignedsegment.pyx", line 2399, in pysam.libcalignedsegment.AlignedSegment.get_tag
  File "pysam/libcalignedsegment.pyx", line 2438, in pysam.libcalignedsegment.AlignedSegment.get_tag
KeyError: "tag 'XI' not present"

No unfortunately STAR does not provide the necessary BAM tags to run through the process

Hi everyone, I was trying to run slam-seq pipeline and here is my command

nextflow run nf-core/slamseq \

-profile singularity \

-c nextflow.config \

--input input.tsv \

--genome GRCm38

But I am getting error:

Unable to execute HTTP request: ngi-igenomes.ngi-igenomes

Any insights on what's going wrong or what am I missing? Any help would be appreciated. Thank you!

Hi <!channel>, I am not being able to figure out what's causing the error here. I was trying to run slam-seq pipeline and here is my command

nextflow run nf-core/slamseq \

-profile singularity \

-c nextflow.config \

--input input.tsv \

--genome GRCm38

But I am getting error:

Unable to execute HTTP request: ngi-igenomes.ngi-igenomes

Any insights on what's going wrong or what am I missing? Any help would be appreciated. Thank you!

Hi, as I understand it, SLAM dunk can only be used for 3' mRNA libraries, is there a pipeline for SLAMseq followed by whole transcriptome sequencing?

Hi Laura. There is nothing like this at the moment. I am working with Niko Popitsch from the Ameres lab on a whole transcriptome solution using STAR. If you like, I can put you in contact

Sorrry for the late follow up but as a potential alternative @Laura Knight, our lab has bam2bakR (which despite it's name can also take fastqs as input), which is a Snakemake pipeline that creates output compatible with the R package I developed call bakR. bakR analyzes SLAM-seq data and performs comparative analyses of SLAM-seq datasets, so it's kind of like DESeq2 for SLAM-seq and similar methods. Manuscript describing bakR is here. @Tobias Neumann bam2bakR uses STAR, but can also use HISAT-3N, which is a bit more appropriate for nucleotide recoding data. Depending on how far along your and Niko's pipeline is, I would love to share notes with you and Niko and potentially collaborate on improving the functionality of either bam2bakR, your pipeline, or both. As someone who has worked extensively with whole transcriptome TimeLapse-seq/SLAM-seq data, I have lots of thoughts 😉

Hi, thanks for making this great pipeline! I'm just curious if there are plans to update/migrate to DSL2, to work with newer nextflow engine versions and be more in alignment with other nf-core pipelines? Thanks!

Hi! Its on my Todo list but that will be a longer term project and I dont yet know when I will have time for it

Hello all. I am trying to run slamseq pipeline but I keep getting errors due to my samplesheet: -[nf-core/slamseq] Pipeline completed with errors- WARN: Graphviz is required to render the execution DAG in the given format -- See http://www.graphviz.org for more info. Error executing process > 'checkDesign (samplesheet.csv)' Caused by: Process

checkDesign (samplesheet.csv)

terminated with an error exit status (1) Command executed: check_design.py samplesheet.csv nfcore_slamseq_design.txt Command exit status: 1 Command output: ERROR: Please check design file header: group,condition,control,reads != group,condition,control,reads Command error: INFO: Environment variable SINGULARITYENV_TMPDIR is set, but APPTAINERENV_TMPDIR is preferred INFO: Environment variable SINGULARITYENV_NXF_DEBUG is set, but APPTAINERENV_NXF_DEBUG is preferred I saved my samplesheet as non-UTF8 version in excel (plain csv) and also generated my samplesheet in nano, but to no avail. Any help will be appreciated. Best wishes.

Hi! This pipeline didn't get a template update with tools 3.0.0 due to a missing

.nf-core.yml

file. Please check the [nf-core/tools documentation](https://nf-co.re/tools) for the correct format. Once you changed that in your

dev

branch you can get a template sync PR by starting the sync GHA action with your pipeline name as an input https://github.com/nf-core/tools/actions/workflows/sync.yml

Hi! I'm trying to run the pipeline, and I'm encoutering a problem running

nf-core/slamseq	v1.0.0

. Despite the documentation saying that it accepts more columns than group, condition, control, reads, https://nf-co.re/slamseq/1.0.0/docs/usage/#--input my run fails when my tsv sample contains a column called name. Does anyone know what the problem may be please? thank you group condition control reads name slz1 4tU 0 JIN8812A11_S103_L005_R1_001.fastq.gz 20_slz1_4tU slz1 4tU 0 JIN8812A11_S103_L006_R1_001.fastq.gz 20_slz1_4tU slz1 DMSO 1 JIN8812A12_S104_L005_R1_001.fastq.gz 20_slz1_DMSO slz1 DMSO 1 JIN8812A12_S104_L006_R1_001.fastq.gz 20_slz1_DMSO