Optionally ignore R1 / R2 after UMI extraction process

[pnatarajan9]

Description of the bug

I have a specific read structure for the paired-end data (R1s carry 12bp UMIs at 3'end; R2s ONLY to be aligned to the transcriptome). The UMI extract steps goes through fine, but then sortmerna fails and when I skip sortmerna step, it fails in STAR alignment - the pipeline is trying to align treating it as paired-end data and R1s will not align in my case.
Post UMI extraction, the data needs to be treated as single-end data (ONLY R2s). At this stage, I would like to run the SortMeRNA (and remove the rRNA reads and get stats), followed by STAR alignment and all the downstream steps including umitools dedup step.
Please help with an example samplesheet.csv, and also the nfcore run command (with specific parameters set in order run UMI-based rnaseq analyses for my specific project).
Thank you very much for your help in advance!
-Padma

Command used and terminal output

-[nf-core/rnaseq] 1/1 samples skipped since they failed STAR 5% mapped threshold:
    0.0%: 01exp3X3MUSxsample01_S0
-
nextflow run rnaseq/nf-core-rnaseq-3.4/workflow \
       --input ./samplesheet.csv \
       -profile singularity \
       -config research.config \
       --with_umi \
       --umitools_extract_method=regex \
       --umitools_bc_pattern='.*(?P<umi_1>.{12})$' \
       --save_umi_intermeds \
       --genome mouse-ensembl-grcm38-r91 \
       --skip_bigwig \
       --remove_ribo_rna
This threw an error with Sortmerna.
Then I removed "--remove_ribo_rna" and re-ran the pipeline.
Now I got: [nf-core/rnaseq] 1/1 samples skipped since they failed STAR 5% mapped threshold:
Error executing process > 'NFCORE_RNASEQ:RNASEQ:QUANTIFY_STAR_SALMON:SALMON_QUANT
The UMI extract step went through fine, but STAR fails.

Relevant files

No response

System information

No response

[drpatelh]

This will be fixed in #771. I have added a new --umi_discard_read parameter which in your case you would set to --umi_discard_read 1. I ended up going with @grst suggestion of filtering the channels after the UMITOOLS_EXTRACT module was run.

Be great if you can test this out @pnatarajan9 when the PR above is merged into dev. Please let me know if you spot any issues.

nextflow pull nf-core/rnaseq
nextflow run nf-core/rnsaeq <OTHER_PARAMETERS> -r dev

[pnatarajan9]

Hi Harshil, Thank you very much for working on the UMI-based RNA-Seq pipeline specific to our experimental setup! I will definitely love to test it as soon as possible. I will get back to you if I have any questions. with best regards, Padma

…

On Mon, Feb 21, 2022 at 3:30 PM Harshil Patel ***@***.***> wrote: This will be fixed in #771 <#771>. I have added a new --umi_discard_read parameter which in your case you would set to --umi_discard_read 1. I ended up going with @grst <https://github.com/grst> suggestion of filtering the channels after the UMITOOLS_EXTRACT module was run. Be great if you can test this out @pnatarajan9 <https://github.com/pnatarajan9> when the PR above is merged into dev. Please let me know if you spot any issues. nextflow pull nf-core/rnaseq nextflow run nf-core/rnsaeq <OTHER_PARAMETERS> -r dev — Reply to this email directly, view it on GitHub <#750 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AEHIZ27A6JHS46BMPJEBO63U4LDJ7ANCNFSM5LUU7O6A> . Triage notifications on the go with GitHub Mobile for iOS <https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android <https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[pnatarajan9]

Hi Harshil, I tried to implement the "dev" version of the nf-core rnaseq pipeline using singularity images. Here are the commands I used: *#------------* *conda create --name nfcore_v2.2_nextflow_v21.10.6 --channel bioconda --channel conda-forge \* * python=3.9.10 nf-core=2.2 nextflow=21.10.6* *#--------* *conda activate nfcore_v2.2_nextflow_v21.10.6* *nf-core download rnaseq -r dev* *#ADD the singularity dir to bashrc* *export NXF_SINGULARITY_CACHEDIR="/nfcorepipelines/singularity_cachedir_dev"* *#--------* I get an error as follows: nf-core/tools version 2.2 In addition to the pipeline code, this tool can download software containers. *?** Download software container images: *singularity If transferring the downloaded files to another system, it can be convenient to have everything compressed in a single file. *This is **not** recommended when downloading Singularity images, as it can take a long time and saves very little space.* *?** Choose compression type: *none INFO Saving 'nf-core/rnaseq' download.py:160 Pipeline revision: 'dev' Pull containers: 'singularity' Using $NXF_SINGULARITY_CACHEDIR': /nfcorepipelines/singularity_cachedir_dev Output directory: 'nf-core-rnaseq-dev' INFO Downloading workflow files from GitHub download.py:163 INFO Downloading centralised configs from GitHub download.py:167 INFO Found *1* container download.py:472 Pulling singularity images ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 0% • 0/1 completed ${ workflow.containerEngine == Copying from cache to target directory ━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ *CRITICAL* *[*Errno *2**]* No such file or directory: 'singularity_cachedir_dev/${ download.py:182 workflow.containerEngine == .img' I get the SAME error while implementing nf-core rnaseq version 3.5 (while version 3.4 implementation worked fine). Thank you for your help in advance! -Padma

…

On Mon, Feb 21, 2022 at 3:30 PM Harshil Patel ***@***.***> wrote: This will be fixed in #771 <#771>. I have added a new --umi_discard_read parameter which in your case you would set to --umi_discard_read 1. I ended up going with @grst <https://github.com/grst> suggestion of filtering the channels after the UMITOOLS_EXTRACT module was run. Be great if you can test this out @pnatarajan9 <https://github.com/pnatarajan9> when the PR above is merged into dev. Please let me know if you spot any issues. nextflow pull nf-core/rnaseq nextflow run nf-core/rnsaeq <OTHER_PARAMETERS> -r dev — Reply to this email directly, view it on GitHub <#750 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AEHIZ27A6JHS46BMPJEBO63U4LDJ7ANCNFSM5LUU7O6A> . Triage notifications on the go with GitHub Mobile for iOS <https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android <https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[drpatelh]

Hi @pnatarajan9 you will need to install the dev version of nf-core/tools. Can you do the following and try again:

conda activate nfcore_v2.2_nextflow_v21.10.6
pip install --upgrade --force-reinstall git+https://github.com/nf-core/tools.git@dev

nf-core download rnaseq -r dev

[pnatarajan9]

Hi Harshil, We have completed the testing on the nf-core RNA-Seq "dev" version and it works well with the changes you have implemented to perform UMI-based RNA-Seq analysis! Thank you and the nf-core team who have contributed for this development. best, Padma

…

On Mon, Feb 21, 2022 at 3:30 PM Harshil Patel ***@***.***> wrote: Closed #750 <#750>. — Reply to this email directly, view it on GitHub <#750 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AEHIZ24OMOZN76EAVSQ5BFDU4LDJ5ANCNFSM5LUU7O6A> . Triage notifications on the go with GitHub Mobile for iOS <https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android <https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>. You are receiving this because you were mentioned.Message ID: ***@***.***>