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Hello, I've been able to run the test run for the ...
# clipseq
l
Hello, I've been able to run the test run for the CLIPseq pipeline. I was wondering, as the pipeline doesn't do the demultiplexing (as far as I understand) if someone could suggest a tool to do that. I've never done it before... Also, how the pipeline treats replicates ? There treated as different sample and we have as many results as we have replicates ? Could someone help me on that please ? Thanks a lot !
c
Hi Li, you can use the tool Ultraplex to do your demultiplexing. You can also try out app.flow.bio which is a free web interface over nextflow pipelines which allows you to do the demultiplexing and has a more recent version of CLIP pipeline. using the dsl1 version, yes you have as many results as replicates. if you fork the branch feat-2-0 you can do replicate merging/more sophisticated stuff but be warned its actively in development in the nf-core repo so will frequently be updated
note here im talking about biological replicates -- if what you mean is technical replicates/resequencing/sequencing split into multiple files then the pipeline can concatenate the FASTQs for you similar to other nextflow pipelines by just giving the files the same sample name
s
Yep, I think usually sequencing core facility does it so it is not standard part of the pipeline.. that said, there is apparently https://nf-co.re/demultiplex/1.6.0 which seems up to date
c
oh nice! i havent tried https://github.com/fulcrumgenomics/fqtk before!
l
Thanks for your answer. What is dsl1 ? What's the difference with the github version ? The version of the software ? How the data is stored in the web interface ? Is it kept afterwards ?
Yes I was talking about biological replicates. Nice, any idea of a timeframe for the version 2 ?
Thanks for the demultiplexing tools. Yes usually the plateform does it but here it will not be standard... so I'll have to do it after taking care of the UMI.
Also, I've got an error in the pipeline (on part of the data used for the paper). Does anyone has already sea this please ?
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Caused by:
 Can't stage file s3:///ngi-igenomes/igenomes/Homo_sapiens/Ensembl/GRCh37/Sequence/STARIndex -- reason: /mnt/beegfs/home/clabbe/stagein/workingDir/stage/2d/656d48dc2a531205f6b5faeb926b08/STARIndex: Communication error on send
c
This is to do with not being able to access the igenomes file. You can use any genomic files you have access to - could be a uni fire wall issue maybe. Timeframe for an official v2 release is realistically a month. Cheers!
DSL1 is an old version of nextflow language. The version on app.flow.bio is also on GitHub so you can check it out - it’s quite similar to what will be the version 2 release on nfcore and afterwards will be replaced by version 2 release on nfcore. Data is securely stored on the ule lab server at kings college London, you have a user account with privacy settings, not even admins can see your data unless you share with them. The data is stored for as long as you like, you can delete it at any time.
l
Thanks a lot for your help. I have another question 😅 I'm not sure I understand the content of the smallRNA file for human. I took a look at the source https://github.com/ulelab/smallRNAs/blob/main/Sources.md and I see that the first file (tRNA) is longer than the smallRNA given with the pipeline and there's almost only tRNA in the file. So I'm wondering, what's really in this file ? How those sequences were choosen please ?
c
oh sorry i see you have the table already - the small RNA files should be the tRNA and rRNA that are given, merged into one file
ignore the snRNA and the rest
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