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Hello!! I am dealing with CRISPRi data, where I h...
# crisprseq
e
Hello!! I am dealing with CRISPRi data, where I have 3 main "cell types" WT. heterozigous, homozigous. 2 timepoints, d0 and d31. And 2 biological replicates. 1. Is there any way of indicating in the pipeline that I have 2 replicates per condition? 2. What we are interested in, is in compare the "progression" (d31vsd0) of the different cell types. So progression of WT vs progression of homozigous, etc. To do so with this pipeline, it can only be done by doing pairwise comparisons within the same cell type (d0 vs d31) and compare then, "manually" the results and outputs extracted between different progressions, right? Thanks a lot guys! Eric
l
Hi Eric, if i understand right you want to do: • WT d31 vs d0 • Homozygous d31 vs d0 • Heterozygous d31 vs d0 and then look at which genes are common and different between these 3 comparisons?
for 1. In the condition column you can add _R1 and _R2
e
Yes! I assume that there is not a direct way of comparing for example (WT d31 vs d0) vs (Homozygous d31 vs d0), so I was just aiming to get this first step, to then manually compare and look for the differences between these "progressions". For 1. I understand then samplesheet.csv have 4 columns. In 'sample' column i was already differentitating each row with its unique identifier (eg. wt_d0_1, wt_d0_2, wt_d31_1,...) should i then do the same in condition? or maybe in sample i have to "group" replicates by including the same label for them? Thanks again!
l
it depends if you want your replicates to be differentiated in the mle/rra process, which gene essentiality are you using? I usually do diffierentiate them in the condition column too
Yes there’s no direct way of comparing them in crisprseq yet, but however you could look into the MAGeCKFlute package : https://www.bioconductor.org/packages/release/bioc/vignettes/MAGeCKFlute/inst/doc/MAGeCKFlute.html#visualization-of-negati[…]and-positive-selections the 9 square plots allow you to visualise different comparisons on the x axis and y axis
e
I belive our idea of replicates was to increase somehow the power of our inference/statistics/conclusions. So i guess if the pipeline merge by accounting that they are replicates it will be enough. We are basically doing this CRISPR on some chr7 genes that were selected specifically. I'll check this plot you are mentioning ! I guess this is something that can be done with the output of the pipeline? Appart from that. After having adjusted these changes in samplesheet.csv and also in contrasts.csv, i get an error when the pipeline jumps into MAGECK TEST: -[nf-core/crisprseq] Pipeline completed with errors- ERROR ~ Error executing process > 'NFCORE_CRISPRSEQCRISPRSEQ SCREENINGMAGECK_TEST (null_null)' Caused by: Process 
NFCORE_CRISPRSEQ:CRISPRSEQ_SCREENING:MAGECK_TEST (null_null)
terminated with an error exit status (255) Command executed: mageck \ test \ \ -t null \ -c null \ -k count_table.count.txt \ -n null_null cat <<-END_VERSIONS > versions.yml "NFCORE_CRISPRSEQCRISPRSEQ SCREENINGMAGECK_TEST": mageck: $(mageck -v) END_VERSIONS Command exit status: 255 Command output: (empty) Command error: INFO: Convert SIF file to sandbox... INFO @ Thu, 14 Nov 2024 155615: Parameters: /usr/local/bin/mageck test -t null -c null -k count_table.count.txt -n null_null INFO @ Thu, 14 Nov 2024 155615: Welcome to MAGeCK v0.5.9.5. Command: test INFO @ Thu, 14 Nov 2024 155615: Loading count table from count_table.count.txt INFO @ Thu, 14 Nov 2024 155615: Processing 1 lines.. INFO @ Thu, 14 Nov 2024 155615: Loaded 2959 records. INFO @ Thu, 14 Nov 2024 155615: Loading Rnw template file: /usr/local/lib/python3.9/site-packages/mageck/test_report.Rmd. INFO @ Thu, 14 Nov 2024 155615: Loading R template file: /usr/local/lib/python3.9/site-packages/mageck/plot_template.RTemplate. INFO @ Thu, 14 Nov 2024 155615: Loading R template file: /usr/local/lib/python3.9/site-packages/mageck/plot_template_indvgene.RTemplate. INFO @ Thu, 14 Nov 2024 155615: Loading Rnw template file: /usr/local/lib/python3.9/site-packages/mageck/plot_template.Rnw. ERROR @ Thu, 14 Nov 2024 155615: Sample label null does not match records in your count table. ERROR @ Thu, 14 Nov 2024 155615: Sample labels in your count table: het_d31_rep1,homo_d0_rep2,homo_d31_rep1,wt_d0_rep1,wt_d31_rep2,homo_d0_rep1,het_d0_rep1,het_d31_rep2,homo_d31_rep2,wt_d31_rep1,het_d0_rep2,wt_d0_rep2 INFO: Cleaning up image... Work dir: /CEPH/users/mdistefano/analysis/CRISPR/crsprseq/work/48/ad81aa7183ca5f82eea2d7ea9043b6 Tip: view the complete command output by changing to the process work dir and entering the command 
cat .command.out
-- Check '.nextflow.log' file for details ERROR ~ Pipeline failed. Please refer to troubleshooting docs: https://nf-co.re/docs/usage/troubleshooting -- Check '.nextflow.log' file for details I guess that it is a format issue, but im not sure where do i have to fix, and what
l
could you please paste the command line you are running along with the contrast file and samplesheet?
e
l
Your contrast file isn’t under the right format it should follow something like this with these specific headers
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The contrast file must contain the headers "reference" and "treatment". These two columns should be separated with a semicolon (;) and contain the csv extension. You can also integrate several samples/conditions by comma-separating them in each column. Please find an example below:
e
ohh, I see, and that is how i indicate to the pipeline that i have biological replicates, by adding them in the same column , right?
l
yes 🙂
e
Thanks a lot !!!! I'll check and review every advise you gave me. 😀
k
Hello! I am actually having the same error, I am not sure what can be the issue:
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ERROR ~ Error executing process > 'NFCORE_CRISPRSEQ:CRISPRSEQ_SCREENING:MAGECK_TEST (moi5_s1,moi5_s2,moi5_s3,,_mock5_s1,mock5_s2,mock5_s3)'

Caused by:
  Process `NFCORE_CRISPRSEQ:CRISPRSEQ_SCREENING:MAGECK_TEST (moi5_s1,moi5_s2,moi5_s3,,_mock5_s1,mock5_s2,mock5_s3)` terminated with an error exit status (255)


Command executed:

  mageck  \
      test \
       \
      -t moi5_s1,moi5_s2,moi5_s3,, \
      -c mock5_s1,mock5_s2,mock5_s3 \
      -k count_table.count.txt \
      -n moi5_s1,moi5_s2,moi5_s3,,_mock5_s1,mock5_s2,mock5_s3
  
  cat <<-END_VERSIONS > versions.yml
  "NFCORE_CRISPRSEQ:CRISPRSEQ_SCREENING:MAGECK_TEST":
      mageck: $(mageck -v)
  END_VERSIONS

Command exit status:
  255

Command output:
  (empty)

Command error:
  INFO  @ Wed, 14 May 2025 15:45:50: Parameters: /usr/local/bin/mageck test -t moi5_s1,moi5_s2,moi5_s3,, -c mock5_s1,mock5_s2,mock5_s3 -k count_table.count.txt -n moi5_s1,moi5_s2,moi5_s3,,_mock5_s1,mock5_s2,mock5_s3 
  INFO  @ Wed, 14 May 2025 15:45:50: Welcome to MAGeCK v0.5.9.5. Command: test 
  INFO  @ Wed, 14 May 2025 15:45:50: Loading count table from count_table.count.txt  
  INFO  @ Wed, 14 May 2025 15:45:50: Processing 1 lines.. 
  INFO  @ Wed, 14 May 2025 15:45:50: Loaded 77441 records. 
  INFO  @ Wed, 14 May 2025 15:45:50: Loading Rnw template file: /usr/local/lib/python3.9/site-packages/mageck/test_report.Rmd. 
  INFO  @ Wed, 14 May 2025 15:45:50: Loading R template file: /usr/local/lib/python3.9/site-packages/mageck/plot_template.RTemplate. 
  INFO  @ Wed, 14 May 2025 15:45:50: Loading R template file: /usr/local/lib/python3.9/site-packages/mageck/plot_template_indvgene.RTemplate. 
  INFO  @ Wed, 14 May 2025 15:45:50: Loading Rnw template file: /usr/local/lib/python3.9/site-packages/mageck/plot_template.Rnw. 
  ERROR @ Wed, 14 May 2025 15:45:50: Sample label moi5_s1 does not match records in your count table. 
  ERROR @ Wed, 14 May 2025 15:45:50: Sample labels in your count table: sample1,sample2,sample3,sample4,sample5,sample6,sample7,sample8,sample9,sample10,sample11,sample12,sample13,sample14,sample15
I am attaching the samplesheet and contrast files
samplesheet_RITM0029948.csvcontrasts.csv
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