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pinellolab / CRISPResso2

Analysis of deep sequencing data for rapid and intuitive interpretation of genome editing experiments
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ERROR: Flash failed to run, please check the log file. #8

Closed bilalSarwar2020 closed 5 years ago

bilalSarwar2020 commented 5 years ago

Hello I am trying to run the CRISPRessoPooled command on the pooled deep sequencing data. i run my script several times as per suggestion, but got an error "ERROR: Flash failed to run, please check the log file" here is the script that i run (base) bilal@ubuntu:~/workshop/pooled samples$ CRISPRessoPooled -r1 6297_1_1.fastq.rar -r2 6297_1_2.fastq.rar -f support_file.txt Flash Package is already installed.

`~CRISPRessoPooled~
-Analysis of CRISPR/Cas9 outcomes from POOLED deep sequencing data- 

           _                                                 _              
          '  )                                              '  )            
          .-'          _______________________              .-'             
         (____         | __  __  __     __ __  |           (____            
      C)|     \        ||__)/  \/  \|  |_ |  \ |        C)|     \           
        \     /        ||   \__/\__/|__|__|__/ |          \     /           
         \___/         |_______________________|           \___/            

                      [CRISPresso version 2.0.27]                           
                [Kendell Clement and Luca Pinello 2019]                     
             [For support contact kclement@mgh.harvard.edu]

INFO @ Sat, 27 Apr 2019 07:20:48: Checking dependencies...

INFO @ Sat, 27 Apr 2019 07:20:48:

All the required dependencies are present!

INFO @ Sat, 27 Apr 2019 07:20:48: Only the Amplicon description file was provided. The analysis will be perfomed using only the provided amplicons sequences.

INFO @ Sat, 27 Apr 2019 07:20:48: Creating Folder CRISPRessoPooled_on_6297_1_1.rar_6297_1_2.rar

WARNING @ Sat, 27 Apr 2019 07:20:48: Folder CRISPRessoPooled_on_6297_1_1.rar_6297_1_2.rar already exists.

INFO @ Sat, 27 Apr 2019 07:20:48: Merging paired sequences with Flash...

CRITICAL @ Sat, 27 Apr 2019 07:20:48:

ERROR: Flash failed to run, please check the log file. `

the log file is attached CRISPRessoPooled_RUNNING_LOG.txt Kindly help me

lucapinello commented 5 years ago

It seems you are using fastq compressed with RAR

6297_1_1.fastq.rar

Only Gzip is supported i.e. files with extensions fastq.gz.

bilalSarwar2020 commented 5 years ago

Dear Lucapinello Thanks for your quick reply. Your Suggestion worked very well for the FLASH issue. after changing the file format, I the script again and it ends with a new error.

ERROR: No reads in input or no reads survived the average or single bp quality filtering.

CRISPResso2 failed CRITICAL @ Sat, 27 Apr 2019 08:03:47: ERROR: CRISPResso amplicon #0 failed

I used Fastqc programme to check the QC of my data.

CRISPRessoPooled_RUNNING_LOG.txt

KIndly help me. I am new in Linux environment

lucapinello commented 5 years ago

Try with the uncompressed file.

On Sat, 27 Apr 2019, 11:33 am bilalSarwar2020, notifications@github.com wrote:

Dear Lucapinello Thanks for your quick reply. Your Suggestion worked very well for the FLASH issue. after changing the file format, I the script again and it ends with a new error.

ERROR: No reads in input or no reads survived the average or single bp quality filtering.

CRISPResso2 failed CRITICAL @ Sat, 27 Apr 2019 08:03:47: ERROR: CRISPResso amplicon #0 failed

I used Fastqc programme to check the QC of my data.

CRISPRessoPooled_RUNNING_LOG.txt https://github.com/pinellolab/CRISPResso2/files/3124017/CRISPRessoPooled_RUNNING_LOG.txt

KIndly help me. I am new in Linux environment

— You are receiving this because you modified the open/close state. Reply to this email directly, view it on GitHub https://github.com/pinellolab/CRISPResso2/issues/8#issuecomment-487295602, or mute the thread https://github.com/notifications/unsubscribe-auth/AAIH72TO4KCT5NGJP6J6FDLPSRW6NANCNFSM4HI4K46A .

bilalSarwar2020 commented 5 years ago

error still exist ERROR: global name 'errno' is not defined CRISPResso2 failed CRITICAL @ Sat, 27 Apr 2019 09:10:59: ERROR: CRISPResso amplicon #0 failed

log file attached CRISPRessoPooled_RUNNING_LOG.txt

lucapinello commented 5 years ago

In order to debug this issue we need to have access to all your input files and the exact command you are using. Can you please share them with us?

On Sat, 27 Apr 2019, 12:16 pm bilalSarwar2020, notifications@github.com wrote:

error still exist ERROR: global name 'errno' is not defined CRISPResso2 failed CRITICAL @ Sat, 27 Apr 2019 09:10:59: ERROR: CRISPResso amplicon #0 failed

log file attached CRISPRessoPooled_RUNNING_LOG.txt https://github.com/pinellolab/CRISPResso2/files/3124070/CRISPRessoPooled_RUNNING_LOG.txt

— You are receiving this because you modified the open/close state. Reply to this email directly, view it on GitHub https://github.com/pinellolab/CRISPResso2/issues/8#issuecomment-487298984, or mute the thread https://github.com/notifications/unsubscribe-auth/AAIH72RRKFA2ZRRPNUVG6ULPSR36RANCNFSM4HI4K46A .

bilalSarwar2020 commented 5 years ago

@lucapinello I have sent you the input data at lpinello@mgh.harvard.edu. thanks for your help

lucapinello commented 5 years ago

The link provided didn't work for me.

Can you share the data with a direct link without authorization?

Thanks,

Luca

On Sun, Apr 28, 2019 at 2:40 AM bilalSarwar2020 notifications@github.com wrote:

@lucapinello https://github.com/lucapinello I have sent you the input data at lpinello@mgh.harvard.edu . thanks for your help

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/pinellolab/CRISPResso2/issues/8#issuecomment-487349893, or mute the thread https://github.com/notifications/unsubscribe-auth/AAIH72XQMO653ICLOFGDLHLPSVBG3ANCNFSM4HI4K46A .

bilalSarwar2020 commented 5 years ago

@lucapinello kindly check your email.

kclem commented 5 years ago

Hi @bilalSarwar2020,

I believe the problem is because the path to your fastqs has a space in it (the "pooled samples" folder).

I just pushed a fix for this problem to github, but you should be able to fix the problem for your current version by changing the folders with spaces in them (e.g. "pooled samples" folder to "pooled_samples") and rerunning it.

Let me know if that works.

bilalSarwar2020 commented 5 years ago

@lucapinello @kclem by adding the --name ONLY_AMPLICONS_XXXX in script solved the issue. it was missing in the previous command. alhamdulillah thanks a lot for your help :)