# Integrating Day 0 Controls in a CRISPR Screen Analysis with Mageck: Seeking Advice

**URL:** <https://help.galaxyproject.org/t/integrating-day-0-controls-in-a-crispr-screen-analysis-with-mageck-seeking-advice/15067>\
**Category:** Uncategorized\
**Tags:** single-cell\
**Created:** [March 21, 2025, 7:54pm UTC](https://help.galaxyproject.org/t/integrating-day-0-controls-in-a-crispr-screen-analysis-with-mageck-seeking-advice/15067 "2025-03-21T19:54:21Z")\
**Posts on this page:** 2\
**Page:** 1

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**Author:** ![jcgonzaleza](https://avatars.discourse-cdn.com/v4/letter/j/cdc98d/32.png) [@jcgonzaleza](https://help.galaxyproject.org/u/jcgonzaleza)\
**Post date:** [March 21, 2025, 7:54pm UTC](https://help.galaxyproject.org/t/integrating-day-0-controls-in-a-crispr-screen-analysis-with-mageck-seeking-advice/15067/1 "2025-03-21T19:54:21Z")

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First of all, thank you very much for your attention. This is my first time participating in a forum of this kind, and any help is greatly appreciated.

I am new to the field of bioinformatics and currently using the Mageck tool (I’ve been working with Mageck count, Mageck test, and Mageck mle) within the Galaxy platform to analyze a single-cell screen experiment I conducted.

My main challenge lies in how to structure the samples (I have extracted the counts from NGS) when performing the corresponding comparisons. Specifically, my experiment has **two genotypes** (WT and KO) that were infected with an sgRNA library. After a positive selection, I collected samples on **day 5**. Additionally, I have two replicates for each experiment in both genotypes, resulting in a total of **8 samples** (see the diagram).

 ![imagen](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/6/69194d3e081cf3cf6ad042ed278baa76ca096e73.png)

My primary interest is identifying which guides are overrepresented in the KO condition compared to the standard WT distribution. I can handle this comparison using Mageck test (RRA) by setting the WT genotype as the control sample.

However, I would like to account for the samples from **day 0** to control for any potential biases in the initial guide distribution between both genotypes, ensuring that they are not mistakenly interpreted as enriched guides in the KO vs. WT comparison.

How could I achieve this initial standardization? Is it possible to do so using Mageck? Is there any other tool that would be better suited for my experimental design?

Thank you very much for your help!

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**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\
**Post date:** [March 21, 2025, 8:07pm UTC](https://help.galaxyproject.org/t/integrating-day-0-controls-in-a-crispr-screen-analysis-with-mageck-seeking-advice/15067/2 "2025-03-21T20:07:56Z")

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Welcome @jcgonzaleza

This is our tutorial for the tools involved. Have you seen this?

> **[Genome Annotation / CRISPR screen analysis / Hands-on: CRISPR screen analysis](https://training.galaxyproject.org/topics/genome-annotation/tutorials/crispr-screen/tutorial.html)**
>
> Genome annotation is a multi-level process that includes prediction of protein-coding genes, as well as other functional genome units such as structural RNAs, tRNAs, small RNAs, pseudogenes, control regions, direct and inverted repeats, insertion...

For advanced usage, following the tool’s original tutorials should be helpful. Tutorial 4 here is covering what you are describing, if I am understanding correctly! Everything should be possible in Galaxy. Please ask if you can’t find an option and we can try to help here.

> **[MAGeCK / Wiki / advanced\_tutorial](https://sourceforge.net/p/mageck/wiki/advanced_tutorial/)**

Hope this helps! 🙂