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hyunhwan-jeong / CB2

CB2 is an R package which provides functions for hit gene identification and quantification of sgRNA (single-guided RNA) abundances for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) pooled screen data analysis. Details are in Jeong et al. (2019) <doi:10.1101/gr.245571.118> and Baggerly et al. (2003) <doi:10.1093/bioinformatics/btg173>.
https://cran.r-project.org/web/packages/CB2/index.html
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Fasta file issue #20

Closed bennychang2 closed 1 year ago

bennychang2 commented 1 year ago

I am trying to use the CRISPRCloud2 platform that you created to do DRISPR analysis. I am encountering issue with uploading of the data of the pooled sgRNA library. I am using a mouse library generated by Weissman Lab which was deposited at Addgene under two subpools: #893987 and #893988. I tried to use the excel file which contains the sequence information as input to upload to the platform, but was not successful. I tried converted it to WORD, to SnapGene, changing the file name, but none was helpful. I got "invalid file selected" error message. Attached is the text file with all sequences in fasta format. Please let me know what needs to be done so that the analysis can move forward. Thank you very much. gRANs-only2.fasta.txt

hyunhwan-jeong commented 1 year ago

Can you try to run this without the .txt extension? I believe that the file name has to be gRANs-only2.fasta

bennychang2 commented 1 year ago

Hi Hyun-Hwan,

Thanks. You are right. It moves forward after I changed the file extension. Thank you very much.

Benny

From: Hyun-Hwan Jeong @.> Sent: Wednesday, July 26, 2023 1:55 PM To: hyunhwan-jeong/CB2 @.> Cc: Chang,Benny @.>; Author @.> Subject: [EXTERNAL] Re: [hyunhwan-jeong/CB2] Fasta file issue (Issue #20)

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Can you try to run this without the .txt extension? I believe that the file name has to be gRANs-only2.fasta

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bennychang2 commented 1 year ago

Hi Hyun-Hwan

I submitted an analysis last Friday, and the data was successfully uploaded. But I used the link to access the results a few times, and I got: Processing The analysis is waiting for its turn or is still going on. Please return to this page later.

Should I continue to wait for it or is there an issue that I should resubmit? Thanks.

Benny

hyunhwan-jeong commented 1 year ago

Please send me the link to @.*** so that I can take a look. Thank you,

On Mon, Jul 31, 2023 at 11:35 AM bennychang2 @.***> wrote:

Hi Hyun-Hwan

I submitted an analysis last Friday, and the data was successfully uploaded. But I used the link to access the results a few times, and I got: Processing The analysis is waiting for its turn or is still going on. Please return to this page later.

Should I continue to wait for it or is there an issue that I should resubmit? Thanks.

Benny

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hyunhwan-jeong commented 1 year ago

@bennychang2 I deleted the link on Github.

bennychang2 commented 1 year ago

Thank you. I guess the link is accessible to everyone. Thank you very much. Let me know whether the analysis is stalled or simply too many requests are being processed. Thanks

hyunhwan-jeong commented 1 year ago

I passed the link to the person who can access the inside, and I will update you once we figure it out.

On Mon, Jul 31, 2023 at 1:43 PM bennychang2 @.***> wrote:

Thank you. I guess the link is accessible to everyone. Thank you very much. Let me know whether the analysis is stalled or simply too many requests are being processed. Thanks

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bennychang2 commented 1 year ago

It's been three days; I wonder if I should submit the job again?

hyunhwan-jeong commented 1 year ago

I haven’t heard back from the team yet, can you try it again? Right now I am out of town and maybe slow to respond until Saturday.

On Thursday, August 3, 2023, bennychang2 @.***> wrote:

It's been three days; I wonder if I should submit the job again?

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hyunhwan-jeong commented 1 year ago

@bennychang2, sorry for the delay, I was able to check the run, and it has been completed analysis. Are you still seeing the same message? If so, I can send the outputs to your email address. Let me know if you want.

bennychang2 commented 1 year ago

It showed that my webserver is ok; cloudfare is ok, but crispr.nrihub is down. So I could not access my results.

hyunhwan-jeong commented 1 year ago

I was told that something is wrong with the cloudfare, but I can send a result to you (zipped file). please send me your email address to @.*** so that I can send a zipped file that contains a result.

Best regards,

On Thu, Aug 10, 2023 at 2:34 PM bennychang2 @.***> wrote:

It showed that my webserver is ok; cloudfare is ok, but crispr.nrihub is down. So I could not access my results.

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bennychang2 commented 1 year ago

bhchang@mdanderson.org

bennychang2 commented 1 year ago

The QC results showed low mappability values 24.84 and 27.06. It does not seem to be good!

hyunhwan-jeong commented 1 year ago

Have you checked FASTQC of the FASTQ files? Make sure that FASTQC doesn't show any red flags on per base sequence quality. It's hard to tell why this is happening since we don't store FASTQ files. If you want me to digging, would it be possible that you to share your FASTQ files with me? If you want me to do this, please send me at @.***, and do not send it to any github related email address for your data privacy.

On Fri, Aug 11, 2023 at 1:12 PM bennychang2 @.***> wrote:

The QC results showed low mappability values 24.84 and 27.06. It does not seem to be good!

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bennychang2 commented 1 year ago

I think I know what went wrong. I was trying to validate the CRISPRi library that I amplified after transforming clone DNA obtained from Addgene. The library was divided in 2 halves. 83987 has one half and 83988 has the other half. In trying to validate I amplified the CRISPRi part of the DNA in the pool of clones and send for NGS. Because there are two pools (two halves), so each pool should have only half of the CRISPRi. But as an input I used the complete CRISPRi library. No wonder the mappabilities were low.

But now I do not know how I can get an idea of how the good the amplified half pool represent the original half library. Do you know what software can allow me to do that? Thanks.