A community effort to collect a curated set of analysis pipelines built using Nextflow.

nf-core

Hi all, my colleague just tried this pipeline and we are troubleshooting as the Mageck MLE doesn't run with negative control sgRNA. It seems like CRISPRcleanR removed the counts of the non-target control sgRNAs and an error showed up. Is it not meant to run with the non-target control after normalisation or is there anything else we can do to resolve the issue?
Really appreciate any help!

Hi <@U04LN6P2E9J>, I am not the main developer of the screening subworkflow but I will try to help :smile: how did you provide the control gRNAs? and how is your design matrix?

Thanks <@U02JS8EFMMJ>. So I have just asked my colleague for their files. The control was supply as a txt file with the seqeunce of each sgRNA per line. Just the sequence and nothing else. For the design matrix it looks like this:
```Samples	baseline	high_high.1_vs_low_low.1
low	1	0
low.1	1	0
high	1	1
high.1	1	1```
They tried to run it without supplying the control sgRNA and it worked. But there is a worry that the non-essential gene controls used in MAGECK MLE may not be suitable. They are looking at the expression level of a target gene instead of viability of the cells. Being non-essential does not mean it doesn't change the expression level of target gene.

Sorry I forgot to attach the actual nextflow command
```nextflow run nf-core/crisprseq -r 2.3.0 -profile xxx \

    --analysis screening \

    --input samplesheet.csv \

    --library brunello_target_sequence.txt \

    --contrasts contrast.csv \

    --crisprcleanr Brunello_Library \

    --rra \

    --mle \

    --hitselection \

    --mle_control_sgrna control-sgRNA.txt \

    --outdir results_crisprseq \

    -resume \

    --email xxxxxx@xxx \

    -with-tower```


Sorry I can't figure out what could be the problem. As I understand, crisprcleanR shouldn't be removing the control sgRNAs

maybe you can try to modify the `min_reads` and/or `min_targeted_genes` parameters, these are currently 30 and 3 by default

specially `min_reads` I think it could have an effect on your problem

<https://github.com/nf-core/crisprseq/blob/0e9f915c4a3c89d02a66ec58e2decbc832323c8b/modules/nf-core/crisprcleanr/normalize/main.nf|this> is the crisprcleanR code in the pipeline, in case you have used it before and want to have a look

Thanks for the info. I read the Mageck MLE tutorial and it didn't include the non target controls in it. I'll do some more digging on how others do similar analysis for Crispr screening on the effect of targeted protein expression level. Appreciate your help!