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pinellolab / CRISPResso2

Analysis of deep sequencing data for rapid and intuitive interpretation of genome editing experiments
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Issue with frameshift mutation analysis. Unexpected error, please check your input. ERROR: 0 #116

Closed tinoco-amanda closed 3 years ago

tinoco-amanda commented 3 years ago

Hello,

I am using CRISPResso2 and I would like to add frameshift analysis. However, whenever I try adding it to the script it encounters an error and I don't get any output. I have tried without adding --coding_seq and it works perfectly fine. The issue only shows when I add --coding_seq to the script. Would you be able to advise me on how to fix the problem. Below is the example of my script as well as the output. I have also tried adding --debug at the end of the script and I pasted the example of the resulting output.

Additionally, I have tried running this script using CRISPRessoBatch, and I seem to only get this problem on some of the samples, not all of them. When I do not include the --coding_seq command, CRISPRessoBatch runs smoothly through all of the samples and returns an output, so I do not think there is a problem with my input files. The problem only occurs when I add --coding_seq. Any advise would be greatly appreciated!

script: CRISPResso --fastq_r1 1_S1_L001_R1_001.fastq.gz --fastq_r2 1_S1_L001_R2_001.fastq.gz --amplicon_name SLC4_1 --amplicon_seq AMPLICON_SEQ --guide_seq GUIDE_SEQ --coding_seq CODING_SEQ --output_folder OUT_FOLDER --quantification_window_size 10 --quantification_window_center -10 --write_detailed_allele_table --plot_window_size 75 --min_average_read_quality 30 --min_single_bp_quality 5 --min_paired_end_reads_overlap 75 --min_bp_quality_or_N 20 --min_frequency_alleles_around_cut_to_plot 0.01 --annotate_wildtype_allele x --ignore_substitutions --debug

result:


        -Analysis of genome editing outcomes from deep sequencing data-         

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                                       '  )                                     
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                                      (____                                     
                                   C)|     \                                    
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                          [CRISPResso version 2.0.45]                           
                    [Kendell Clement and Luca Pinello 2020]                     
                 [For support contact kclement@mgh.harvard.edu]                 

INFO  @ Fri, 23 Jul 2021 17:35:54:
     Creating Folder /mnt/sequence/atinoco/Miseq/SLC4/troubleshooting/guide3_bash5/errortest_S1.1/CRISPResso_on_1_S1_L001_R1_001_1_S1_L001_R2_001 

INFO  @ Fri, 23 Jul 2021 17:35:54:
     Filtering reads with average bp quality < 30 and single bp quality < 5 and replacing bases with quality < 20 with N ... 

Completed in 15 seconds

INFO  @ Fri, 23 Jul 2021 17:36:10:
     Estimating average read length... 

INFO  @ Fri, 23 Jul 2021 17:36:10:
     Checking average read length from /mnt/sequence/atinoco/Miseq/SLC4/troubleshooting/guide3_bash5/errortest_S1.1/CRISPResso_on_1_S1_L001_R1_001_1_S1_L001_R2_001/1_S1_L001_R1_001_filtered.fastq.gz 

INFO  @ Fri, 23 Jul 2021 17:36:10:
     Average read length is 301 from /mnt/sequence/atinoco/Miseq/SLC4/troubleshooting/guide3_bash5/errortest_S1.1/CRISPResso_on_1_S1_L001_R1_001_1_S1_L001_R2_001/1_S1_L001_R1_001_filtered.fastq.gz 

INFO  @ Fri, 23 Jul 2021 17:36:10:
     Merging paired sequences with Flash... 

INFO  @ Fri, 23 Jul 2021 17:36:10:
     Running FLASH command: flash "/mnt/sequence/atinoco/Miseq/SLC4/troubleshooting/guide3_bash5/errortest_S1.1/CRISPResso_on_1_S1_L001_R1_001_1_S1_L001_R2_001/1_S1_L001_R1_001_filtered.fastq.gz" "/mnt/sequence/atinoco/Miseq/SLC4/troubleshooting/guide3_bash5/errortest_S1.1/CRISPResso_on_1_S1_L001_R1_001_1_S1_L001_R2_001/1_S1_L001_R2_001_filtered.fastq.gz" --min-overlap 75 --max-overlap 100 --allow-outies -z -d /mnt/sequence/atinoco/Miseq/SLC4/troubleshooting/guide3_bash5/errortest_S1.1/CRISPResso_on_1_S1_L001_R1_001_1_S1_L001_R2_001 -o out >>/mnt/sequence/atinoco/Miseq/SLC4/troubleshooting/guide3_bash5/errortest_S1.1/CRISPResso_on_1_S1_L001_R1_001_1_S1_L001_R2_001/CRISPResso_RUNNING_LOG.txt 2>&1 

INFO  @ Fri, 23 Jul 2021 17:36:13:
     Done! 

INFO  @ Fri, 23 Jul 2021 17:36:13:
     Aligning sequences... 

INFO  @ Fri, 23 Jul 2021 17:36:13:
     Processing reads; N_TOT_READS: 0 N_COMPUTED_ALN: 0 N_CACHED_ALN: 0 N_COMPUTED_NOTALN: 0 N_CACHED_NOTALN: 0 

INFO  @ Fri, 23 Jul 2021 17:36:21:
     Processing reads; N_TOT_READS: 10000 N_COMPUTED_ALN: 366 N_CACHED_ALN: 991 N_COMPUTED_NOTALN: 1570 N_CACHED_NOTALN: 7073 

INFO  @ Fri, 23 Jul 2021 17:36:27:
     Processing reads; N_TOT_READS: 20000 N_COMPUTED_ALN: 717 N_CACHED_ALN: 2060 N_COMPUTED_NOTALN: 2873 N_CACHED_NOTALN: 14350 

INFO  @ Fri, 23 Jul 2021 17:36:35:
     Processing reads; N_TOT_READS: 30000 N_COMPUTED_ALN: 1161 N_CACHED_ALN: 2979 N_COMPUTED_NOTALN: 4493 N_CACHED_NOTALN: 21367 

INFO  @ Fri, 23 Jul 2021 17:36:43:
     Processing reads; N_TOT_READS: 40000 N_COMPUTED_ALN: 1574 N_CACHED_ALN: 3963 N_COMPUTED_NOTALN: 6151 N_CACHED_NOTALN: 28312 

INFO  @ Fri, 23 Jul 2021 17:36:43:
     Finished reads; N_TOT_READS: 40184 N_COMPUTED_ALN: 1585 N_CACHED_ALN: 3980 N_COMPUTED_NOTALN: 6180 N_CACHED_NOTALN: 28439 

INFO  @ Fri, 23 Jul 2021 17:36:43:
     Done! 

INFO  @ Fri, 23 Jul 2021 17:36:43:
     Quantifying indels/substitutions... 

INFO  @ Fri, 23 Jul 2021 17:36:44:
     Done! 

INFO  @ Fri, 23 Jul 2021 17:36:44:
     Calculating allele frequencies... 

INFO  @ Fri, 23 Jul 2021 17:36:44:
     Done! 

INFO  @ Fri, 23 Jul 2021 17:36:44:
     Saving processed data... 

INFO  @ Fri, 23 Jul 2021 17:36:44:
     Making Plots... 

Traceback (most recent call last):
  File "/mnt/sequence/atinoco/miniconda2/envs/CRISPResso2/lib/python2.7/site-packages/CRISPResso2/CRISPRessoCORE.py", line 4119, in main
    crispresso2_info['refs'][ref_name]['plot_6_caption'] = "Figure 6: Frameshift and in-frame mutagenesis profiles indicating position affected by modification. The y axis shows the number of reads and percentage of all reads in that category (frameshifted (top) or in-frame (bottom)). %d reads with no length modifications are not shown."%hists_inframe[ref_name][0]
KeyError: 0
CRITICAL @ Fri, 23 Jul 2021 17:37:07:
     Traceback (most recent call last):
  File "/mnt/sequence/atinoco/miniconda2/envs/CRISPResso2/lib/python2.7/site-packages/CRISPResso2/CRISPRessoCORE.py", line 4119, in main
    crispresso2_info['refs'][ref_name]['plot_6_caption'] = "Figure 6: Frameshift and in-frame mutagenesis profiles indicating position affected by modification. The y axis shows the number of reads and percentage of all reads in that category (frameshifted (top) or in-frame (bottom)). %d reads with no length modifications are not shown."%hists_inframe[ref_name][0]
KeyError: 0

CRITICAL @ Fri, 23 Jul 2021 17:37:07:
     Unexpected error, please check your input.

ERROR: 0 

Thank you! 
Amanda
kclem commented 3 years ago

Hi @tinoco-amanda Thanks for using CRISPResso!

I believe this error was fixed a few commits ago -- would you mind updating (current version is 2.1.3) and seeing if it's fixed?

tinoco-amanda commented 3 years ago

Thank you so much! That fixed the issue. I really appreciate the fast response! I also really appreciate the creation of CRISPResso, it is incredibly helpful!

I had one quick question with regards to the frameshift mutation summary data. Is there a way of getting that information added to the summary CRISPRessoBatch_quantification_of_editing_frequency.txt or as a separate output but in summary format so as to not have to go into every individual file to retrieve the data?

Thanks again for your help! I really appreciate it!

Best, Amanda

On Fri, Jul 23, 2021 at 7:59 PM Kendell Clement @.***> wrote:

Hi @tinoco-amanda https://github.com/tinoco-amanda Thanks for using CRISPResso!

I believe this error was fixed a few commits ago -- would you mind updating (current version is 2.1.3) and seeing if it's fixed?

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/pinellolab/CRISPResso2/issues/116#issuecomment-885966960, or unsubscribe https://github.com/notifications/unsubscribe-auth/AU6PGBCRTYKV46SLJL6VH43TZH67PANCNFSM5A4YHNPQ .

kclem commented 3 years ago

I'm glad you find it useful!

Yes, I'll add this to the things in the next release -- I'll make a 'CRISPRessBatch_quantifiction_of_frameshift.txt' that aggregates this.

tinoco-amanda commented 3 years ago

Thank you so much!! Out of curiosity, do you have an idea as to when you will be making the release?

Again, CRISPResso is a great module and I really appreciate you making it. It is great!

Best, Amanda

On Tue, Jul 27, 2021 at 9:40 AM Kendell Clement @.***> wrote:

I'm glad you find it useful!

Yes, I'll add this to the things in the next release -- I'll make a 'CRISPRessBatch_quantifiction_of_frameshift.txt' that aggregates this.

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/pinellolab/CRISPResso2/issues/116#issuecomment-887522589, or unsubscribe https://github.com/notifications/unsubscribe-auth/AU6PGBB66PENMA2HXLMEQKLTZ2ZORANCNFSM5A4YHNPQ .