This error is The specified file 'allele_specific.fastq.gz' cannot be opened.

[yunhu-HY]

We installed CRISPResso2 using anaconda, but when we ran the CRISPResso Examples you provided (CRISPResso --fastq_r1 allele_specific.fastq.gz --amplicon_seq CGAGAGCCGCAGCCATGAACGGCACAGAGGGCCCCAATTTTTATGTGCCCTTCTCCAACGTCACAGGCGTGGTGCGGAGCCACTTCGAGCAGCCGCAGTACTACCTGGCGGAACCATGGCAGTTCTCCATGCTGGCAGCGTACATGTTCCTGCTCATCGTGCTGGG,CGAGAGCCGCAGCCATGAACGGCACAGAGGGCCCCAATTTTTATGTGCCCTTCTCCAACGTCACAGGCGTGGTGCGGAGCCCCTTCGAGCAGCCGCAGTACTACCTGGCGGAACCATGGCAGTTCTCCATGCTGGCAGCGTACATGTTCCTGCTCATCGTGCTGGG --amplicon_name P23H,WT --guide_seq GTGCGGAGCCACTTCGAGCAGC),
we got an error. This error is The specified file 'allele_specific.fastq.gz' cannot be opened.
Any ideas?
Thanks!
YunHu

[Colelyman]

Hi @yunhu-HY,

Thanks for trying CRISPResso and sorry to hear that you can't get the example running. I believe there is an error on the documentation site preventing the download of the allele_specific.fastq.gz file. Can you try redownloading it at this link? http://crispresso.pinellolab.partners.org/static/demo/allele_specific.fastq.gz

[Colelyman]

Yes, that would also cause the issue. If you downloaded the file to your Downloads folder (which is `~/Downloads` on most Mac and Linux machines) then you will need to change to that directory before running CRISPResso. You can do that by running the command `cd ~/Downloads`. You can also see which directory you are in by running the command `pwd`. Good luck!

…

On Aug 13, 2024, at 9:51 AM, yunhu-HY ***@***.***> wrote: Hi, @Colelyman <https://github.com/Colelyman>. Thanks you for your reply. I re-downloaded the file you sent, but I encountered the same error. Could it be that the file I downloaded is not placed in the specified directory? As I am a beginner in bioinformatics, I’m not sure where the problem lies. Could you please help me check if there are any issues with the software I installed and the code I ran? There are software and code that I used. <applewebdata://85EE8ADF-6971-44F6-94C8-1BE6556438D9> — Reply to this email directly, view it on GitHub <#471 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AAIZ3ZNDMUMUCJQPIK5FT7TZRITPDAVCNFSM6AAAAABMMGGFO6VHI2DSMVQWIX3LMV43URDJONRXK43TNFXW4Q3PNVWWK3TUHMYTAMZSG44DCMI>. You are receiving this because you were mentioned.

[yunhu-HY]

I have solved this error. Thank you so much!!!!

[yunhu-HY]

Hello @Colelyman, Can this CRISPResso2 be used to analyze the efficiency of Prime Editing for multiple mutations(the length of the RT template is 50nt ) and insertion and deletion of about 40nt nucleotides? If possible, could you please provide a command? Thanks!

[kclem]

Hi @yunhu-HY, the command would look something like this:
CRISPResso -r1 {fastq_r1} --amplicon_seq {wt_seq},{ins_seq},{del_seq} --amplicon_name WT,INS,DEL

The --amplicon_seq parameter is used to provide the sequences of the expected outcomes. For example, --amplicon_seq AATTTAA,AATGGGGTTAA,AATAA.

If would like to try CRISPResso in prime editing mode, you can provide the extension sequence and other parts from your pegRNA like this:
CRISPResso --fastq_r1 prime_editor.fastq.gz --amplicon_seq ACGTCTCATATGCCCCTTGGCAGTCATCTTAGTCATTACCTGAGGTGTTCGTTGTAACTCATATAAACTGAGTTCCCATGTTTTGCTTAATGGTTGAGTTCCGTTTGTCTGCACAGCCTGAGACATTGCTGGAAATAAAGAAGAGAGAAAAACAATTTTAGTATTTGGAAGGGAAGTGCTATGGTCTGAATGTATGTGTCCCACCAAAATTCCTACGT --prime_editing_pegRNA_spacer_seq GTCATCTTAGTCATTACCTG --prime_editing_pegRNA_extension_seq AACGAACACCTCATGTAATGACTAAGATG --prime_editing_nicking_guide_seq CTCAACCATTAAGCAAAACAT --prime_editing_pegRNA_scaffold_seq GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC
You can try this example with the sequencing file here: prime_editor.fastq.gz