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lucapinello / CRISPResso

Software pipeline for the analysis of CRISPR-Cas9 genome editing outcomes from sequencing data
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Last stage error Error with FLASH for CRISPRessoPooled.py #51

Closed bilalSarwar2020 closed 5 years ago

bilalSarwar2020 commented 5 years ago

Dear Luca Pinello I am using CRISPRessoPooled.py script to analyzed the paired ends reads data by Illumina. the read length is 150 bp. the amplicon size is 180bp long. below is the command that I am used several time. it ends up with an error at last of the analysis I am new to the Linux environment. Kindly help me to resolve this issue. thanks
ERROR: Flash failed to run, please check the log file.

$ python CRISPRessoPooled.py -r1 /home/bilal/Sir_Qayyum_data/6297_1_1.fastq.gz -r2 /home/bilal/Sir_Qayyum_data/6297_1_2.fastq.gz -f /home/bilal/Sir_Qayyum_data/CRISPResso-master/cat.csv 


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

              )                                            )
             (           _______________________          (
            __)__       | __  __  __     __ __  |        __)__
         C\|     \      ||__)/  \/  \|  |_ |  \ |     C\|     \
           \     /      ||   \__/\__/|__|__|__/ |       \     /
            \___/       |_______________________|        \___/

[Luca Pinello 2015, send bugs, suggestions or *green coffee* to lucapinello AT gmail DOT com]

Version 1.0.13

INFO  @ Tue, 05 Feb 2019 22:43:54:
     Checking dependencies... 

INFO  @ Tue, 05 Feb 2019 22:43:54:

 All the required dependencies are present! 

INFO  @ Tue, 05 Feb 2019 22:43:54:
     Only the Amplicon description file was provided. The analysis will be perfomed using only the provided amplicons sequences. 

INFO  @ Tue, 05 Feb 2019 22:43:54:
     Creating Folder CRISPRessoPooled_on_6297_1_1_6297_1_2 

WARNING @ Tue, 05 Feb 2019 22:43:54:
     Folder CRISPRessoPooled_on_6297_1_1_6297_1_2 already exists. 

INFO  @ Tue, 05 Feb 2019 22:43:54:
     Merging paired sequences with Flash... 

CRITICAL @ Tue, 05 Feb 2019 22:43:54:

ERROR: Flash failed to run, please check the log file.
lucapinello commented 5 years ago

Can you send us the log file?

Also can you please try with the new version of CRISPREsso available here: https://github.com/pinellolab/crispresso2

On Tue, Feb 5, 2019 at 12:59 PM bilalSarwar2020 notifications@github.com wrote:

Dear Luca Pinello I am using CRISPRessoPooled.py script to analyzed the paired ends reads data by Illumina. the read length is 150 bp. the amplicon size is 180bp long. below is the command that I am used several time. it ends up with an error at last of the analysis I am new to the Linux environment. Kindly help me to resolve this issue. thanks ERROR: Flash failed to run, please check the log file.

$ python CRISPRessoPooled.py -r1 /home/bilal/Sir_Qayyum_data/6297_1_1.fastq.gz -r2 /home/bilal/Sir_Qayyum_data/6297_1_2.fastq.gz -f /home/bilal/Sir_Qayyum_data/CRISPResso-master/cat.csv


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

          )                                            )
         (           _______________________          (
        __)__       | __  __  __     __ __  |        __)__
     C\|     \      ||__)/  \/  \|  |_ |  \ |     C\|     \
       \     /      ||   \__/\__/|__|__|__/ |       \     /
        \___/       |_______________________|        \___/

[Luca Pinello 2015, send bugs, suggestions or *green coffee* to
lucapinello AT gmail DOT com]

Version 1.0.13

INFO @ Tue, 05 Feb 2019 22:43:54:
Checking dependencies...

INFO @ Tue, 05 Feb 2019 22:43:54:

All the required dependencies are present!

INFO @ Tue, 05 Feb 2019 22:43:54:
Only the Amplicon description file was provided. The analysis will be
perfomed using only the provided amplicons sequences.

INFO @ Tue, 05 Feb 2019 22:43:54:
Creating Folder CRISPRessoPooled_on_6297_1_1_6297_1_2

WARNING @ Tue, 05 Feb 2019 22:43:54:
Folder CRISPRessoPooled_on_6297_1_1_6297_1_2 already exists.

INFO @ Tue, 05 Feb 2019 22:43:54:
Merging paired sequences with Flash...

CRITICAL @ Tue, 05 Feb 2019 22:43:54:

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

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bilalSarwar2020 commented 5 years ago

Dear Luca Pinello Thanks for getting me back. Probably the FLASH was not at the right PATH. I got a clue from your response to issue #49 and after fixing it run successfuly but with the empty output files in the result folder. i attached all the files including the log files. kindly help me

Regards stressed

. CRISPRessoPooled_RUNNING_LOG.txt MAPPING_STATISTICS.txt REPORT_READS_ALIGNED_TO_AMPLICONS.txt SAMPLES_QUANTIFICATION_SUMMARY.txt

lucapinello commented 5 years ago

Do you have bowtie2 installed and in the path?

On Wed, Feb 6, 2019 at 10:50 AM bilalSarwar2020 notifications@github.com wrote:

Dear Luca Pinello Thanks for getting me back. Probably the FLASH was not at the right PATH. I got a clue from your response to issue #49 https://github.com/lucapinello/CRISPResso/issues/49 and after fixing it run successfuly but with the empty output files in the result folder. i attached all the files including the log files. kindly help me

Regards stressed

. CRISPRessoPooled_RUNNING_LOG.txt https://github.com/lucapinello/CRISPResso/files/2837017/CRISPRessoPooled_RUNNING_LOG.txt MAPPING_STATISTICS.txt https://github.com/lucapinello/CRISPResso/files/2837018/MAPPING_STATISTICS.txt REPORT_READS_ALIGNED_TO_AMPLICONS.txt https://github.com/lucapinello/CRISPResso/files/2837019/REPORT_READS_ALIGNED_TO_AMPLICONS.txt SAMPLES_QUANTIFICATION_SUMMARY.txt https://github.com/lucapinello/CRISPResso/files/2837020/SAMPLES_QUANTIFICATION_SUMMARY.txt

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bilalSarwar2020 commented 5 years ago

YES Version 2.2.6 installed

bilal@Plant-Genomics:~$ bowtie2 --version /usr/bin/bowtie2-align-s version 2.2.6 64-bit Built on lgw01-12 Mon Dec 28 11:09:46 UTC 2015 Compiler: gcc version 5.3.1 20151219 (Ubuntu 5.3.1-4ubuntu1) Options: -O3 -m64 -msse2 -funroll-loops -g3 -Wl,-Bsymbolic-functions -Wl,-z,relro -DPOPCNT_CAPABILITY -DWITH_TBB Sizeof {int, long, long long, void*, size_t, off_t}: {4, 8, 8, 8, 8, 8}

lucapinello commented 5 years ago

Can you please try with the new version of CRISPResso and install it via conda?

See the instructions here: https://github.com/pinellolab/crispresso2

On Wed, Feb 6, 2019 at 11:06 AM bilalSarwar2020 notifications@github.com wrote:

YES Version 2.2.6 installed

bilal@Plant-Genomics:~$ bowtie2 --version /usr/bin/bowtie2-align-s version 2.2.6 64-bit Built on lgw01-12 Mon Dec 28 11:09:46 UTC 2015 Compiler: gcc version 5.3.1 20151219 (Ubuntu 5.3.1-4ubuntu1) Options: -O3 -m64 -msse2 -funroll-loops -g3 -Wl,-Bsymbolic-functions -Wl,-z,relro -DPOPCNT_CAPABILITY -DWITH_TBB Sizeof {int, long, long long, void*, size_t, off_t}: {4, 8, 8, 8, 8, 8}

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