Crispr-cas9 screen analysis mageck

Question

[11yj3312]

Hi friends! Now I have a batch of paired-end sequencing data，I don't know whether mageck can handle paired-end sequencing data,I don't find appropriate parameters.What do you do with paired-end sequencing data？ I try just use R1 sequencing data mapped with sgRNA library,but only about 60% reads can be mapped.What is the probable cause?How can I improve it?

[Carambakaracho]

Hi there, I'm not a MAGeCK expert, but I find it difficult to understand what you're trying to achieve. I encourage you to improve your title (e.g. include MAGeCK and PE) to help the right people find your post

Answer 1

[dsull]

I was having the same question as the original poster, and although this is an old question, I wanted to post an answer:

MAGeCK now has paired-end read support: https://bitbucket.org/liulab/mageck/commits/3e3344e64f0dc2d5b20f5895a9c995b5a9cd0124

You use the --fastq-2 option, as follows:

mageck count -l library.txt -n prefix --sample-label A,B,C,D --fastq A_1.fq.gz B_1.fq.gz C_1.fq.gz D_1.fq.gz --fastq-2 A_2.fq.gz B_2.fq.gz C_2.fq.gz D_2.fq.gz

In the example above, we have four samples: A, B, C, D, each of which are associated with a pair of fastq files (for paired-end reads). For example, sample A has A_1.fq.gz and A_2.fq.gz associated with it. The order of the files in the --fastq-2 option should be consistent with the order in the --fastq option. The way the algorithm works (or at least my understanding of it): iff the first read pair doesn't map successfully, then it will try mapping the second read pair.

(Note: The above applies to the current MAGeCK version: 0.5.9.2).

[santos22903]

This will also be a comment to an old question, but I did not find a better place and authors do not seem to reply anywhere. I actually used the --fastq-2 option, the program did not complain, but the "countsummary" file looked like there were no R2 files given. As an example, assuming I have sample1_R1.fq, sample1_R2.fq, sample2_R1.fq, sample2_R2.fq, I ran mageck count with --fastq sample1_R1.fq sample2_R1.fq --fastq-2 sample1_R2.fq sample2_R2.fq arguments. The "countsummary" file has only 2 lines:

sample1_R1.fq label counts etc

sample2_R1.fq label counts etc

Is this what it is supposed to look like? Looks like R2's are "forgotten"

[dsull]

Are the results the same whether you use R1 or R1+R2?

In any case, you should only get one count because of the algorithm I described above.