GeCKO Sequencing Data and Analysis?

[Diana H]

Hello!

I've done my GeCKO screen and very excitingly have received my sequencing data back(!!)--with the caveat of now only having some familiarity with the linux command line and only a cursory idea as to how to convert the raw FASTQ data into a trimmed sgRNA for alignment and counting against the reference library. I'd like to learn how to do this analysis myself. Is there an established workflow with how to handle this data? The papers reference the Hannon lab's FASTX tool but I haven't been able to find a "beginner's level" resource as to how to use this. From here, is this output then piped into Bowtie and aligned (or just counted for number of unique instances?)? And finally, can you then take the Bowtied SAM file and pump that into MaGeCK for analysis? Any help would be thoroughly appreciated. Thank you!

-D

[Alex J]

Hi Diana,

In case you are familiar with R and the package edgeR, I found this paper extremely helpful for analysis of GeCKO data: http://f1000research.com/articles/3-95/v2

The R code that they use for data analysis is explained and annotated in detail here (p. 31ff.): http://bioinf.wehi.edu.au/shRNAseq/pooledScreenAnalysis.pdf

Best,

Alex

[Wei Li]

Hi Diana,

MAGeCK can do all the sequence counting, sgRNA and gene level analysis for you. You can either start from the fastq file, or start from the read count tables you collected. The MAGeCK tutorial gives you a simple workflow to do all the things:

https://sourceforge.net/p/mageck/wiki/demo/

See the detailed instructions from the 3rd demo. If you already have the read counts, you can start from the first demo. 

We are improving the MAGeCK software constantly to make it easy to use for biologists. Let me know if you have further questions! 

[Ben Boward]

Hi,

Is it possible to perform a read count and alignment to a reference gRNA library using MAGeCK from raw fastq or fasta files? 

 

Thanks,

Ben

[Wei Li]

Yes, MAGeCK has a module called "count" that can generate counts from fastq files. See the manual (https://sourceforge.net/p/mageck/wiki/Home/) for more details.

[Diana H]

Wei,

Thank you again for your responses. I've gotten the demos to work and I'm ready for real fastq data! But I now have a very basic question: Do you move the files into the mageck directory in order for mageck to "see" the files? I take it you cannot simply run mageck from any place necessarily for the two to communicate? Also, does it support other input formats (.fasta/.fastqsanger/.txt)? Thank you so much for all your work!

Diana

[Wei Li]

Hi,

In most cases after installation, you can run mageck anywhere so you don't need to move the files to mageck directory. Just type "mageck" in the terminal to test this. Sometimes you need to set up the environment variable, as indicated in the manual:

https://sourceforge.net/p/mageck/wiki/install/

Let me know if you have any questions!

[ZJ 09]

Hi Wei li

Is MAGeCK outperforms the RIGER method, since Julia Joung, 2017 used count_spacer.py and RIGER to analyze the CRISPR activation screen data. 

Thanks

Jie

[Julia Joung]

Hi Jie,

I would recommend using MAGeCK over RIGER - we used RIGER for analyzing CRISPR activation screens before MAGeCK was available.

Best,

Julia

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[Wei Li]

Thanks Julia for the recommendation! We are continuing working on updating MAGeCK software. 

On Saturday, November 2, 2019 at 4:40:06 PM UTC-4, Julia Joung wrote:

Hi Jie,

I would recommend using MAGeCK over RIGER - we used RIGER for analyzing CRISPR activation screens before MAGeCK was available.

Best,

Julia

On Thu, Oct 31, 2019 at 8:47 AM ZJ 09 <zjre...@gmail.com> wrote:

Hi Wei li

Is MAGeCK outperforms the RIGER method, since Julia Joung, 2017 used count_spacer.py and RIGER to analyze the CRISPR activation screen data. 

Thanks

Jie

On Sunday, May 24, 2015 at 7:10:51 AM UTC+8, Wei Li wrote:

Yes, MAGeCK has a module called "count" that can generate counts from fastq files. See the manual (https://sourceforge.net/p/mageck/wiki/Home/) for more details.

On Saturday, May 23, 2015 at 4:30:36 PM UTC-4, Ben Boward wrote:

Hi,

Is it possible to perform a read count and alignment to a reference gRNA library using MAGeCK from raw fastq or fasta files? 

 

Thanks,

Ben

On Wednesday, May 20, 2015 at 8:53:12 PM UTC-4, Wei Li wrote:

Hi Diana,

MAGeCK can do all the sequence counting, sgRNA and gene level analysis for you. You can either start from the fastq file, or start from the read count tables you collected. The MAGeCK tutorial gives you a simple workflow to do all the things:

https://sourceforge.net/p/mageck/wiki/demo/

See the detailed instructions from the 3rd demo. If you already have the read counts, you can start from the first demo. 

We are improving the MAGeCK software constantly to make it easy to use for biologists. Let me know if you have further questions! 

On Wednesday, May 6, 2015 at 9:35:16 PM UTC-4, Diana H wrote:

Hello!

I've done my GeCKO screen and very excitingly have received my sequencing data back(!!)--with the caveat of now only having some familiarity with the linux command line and only a cursory idea as to how to convert the raw FASTQ data into a trimmed sgRNA for alignment and counting against the reference library. I'd like to learn how to do this analysis myself. Is there an established workflow with how to handle this data? The papers reference the Hannon lab's FASTX tool but I haven't been able to find a "beginner's level" resource as to how to use this. From here, is this output then piped into Bowtie and aligned (or just counted for number of unique instances?)? And finally, can you then take the Bowtied SAM file and pump that into MaGeCK for analysis? Any help would be thoroughly appreciated. Thank you!

-D

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[Wei Li]

We used MAGeCK for datasets with no replicates before. However, the results depend on how noisy the data would be. So go ahead and try it.

On Wednesday, November 6, 2019 at 9:00:55 AM UTC-5, Jie wrote:

In the paper, "MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens" the section "Mean-variance modeling" said the number of replicates is usually limited (for example, if we have not replicates), the modeling can be inferred using the mean and variance values of all sgRNAs. However, in the following section "sgRNA test and ranking", it said "Consequently, if there are no replicates, MAGeCK may be less sensitive as it overestimates the variance in one condition".

 

The following is our screen method and we have no replicates, in this case, can we use MAGeCK for the analysis. Is there any relevant references or protocols for this type of screen?

 

"

In our study,  we are conducting a CRISPR-screen followed by using antibody staining intracellular protein and FACS sorting, the method of which is similar to this paper "Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells, Science, 2018". In this paper their screen resulted  in HbF- group  and HbF+ group (see the attached figure), and the read counts were calculated for each individual sgRNA and normalized to total read counts.

[Ryan Ren]

Thanks for your kind reply

Wei Li <li.dav...@gmail.com>于2019年11月8日 周五下午8:37写道：

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