ERROR: cannot access local variable 'new_variant'

[Gabelberg]

I am using CRISPresso2 in batch mode, and with any setting it give me an error and the instruction to try a run with CRISPResso and a seris of parameters. I executed it and this is the error that I receive:
"Traceback (most recent call last):
File "/home/giovanni/miniforge3/envs/crispresso2_env/lib/python3.12/site-packages/CRISPResso2/CRISPRessoCORE.py", line 3500, in main aln_stats = process_single_fastq_write_bam_out(processed_output_filename, crispresso2_info['bam_output'], bam_header, variantCache, ref_names, refs, args, files_to_remove, OUTPUT_DIRECTORY)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/giovanni/miniforge3/envs/crispresso2_env/lib/python3.12/site-packages/CRISPResso2/CRISPRessoCORE.py", line 2039, in process_single_fastq_write_bam_out " ALN_SCORES=" + ('&'.join([str(x) for x in new_variant['aln_scores']])) +\
^^^^^^^^^^^
UnboundLocalError: cannot access local variable 'new_variant' where it is not associated with a value
CRITICAL @ Thu, 25 Sep 2025 12:00:09 (7.0% done):
Unexpected error, please check your input.
ERROR: cannot access local variable 'new_variant' where it is not associated with a value "

What I am missing?
My amplicon and SGDNA are the following:
Amplicon
"ATATAAAGAAAAGAAAAGGGGAGAGGATGGGTCGAATCCCATGTTGCGAAAAGGAGAGTGTGAAGAAGGGTCAGTGGACCCCTGAAGAAGATCATAAGCTGTCTTCCTACATCGCTCAACATGGGACTCGTAATTGGCGCCTCATTCCCAAAAATGCAGGCCTCCAAAGATGTGGGAAGAGCTGTAGGTTACGCTGGACTAACTACCTACGCCCTGATTTAAAACATGGACAGTTCTCCGATGCTGAAGAACAAATCATTGTCAGGCTACATTCCGTTCTAGGCAACAGGTATAAATAACACATAAAATGTATGCATATGTATGTATGTATGTGCATGTATGTGTGTGTGTATCCATTCTCATGACCAAACTCTGAGAGATTGTATCTATACAATGCAAGTACAGGTGGTCCGTGATAGCGGCACAGCTGCCAGGACGAACAGACAATGATGTGAAGAACCATTGGAACACGAAACTGAAAAAGAAGCTGTCAGGTATGGGAATCGATCCCGTTACGCATAAGCCTTACTCCCACCTCATGGCGGAGATAGCCACCACTCTCGCTCCTCCACAGGTGGCTAACCTCGCAGAAGCAACTCTGGGATGTTTGAAAGATGAGATGCTTCATCTTCTAACCAAGAAGCACATAGATATTCAATTCCAATCCCCTGCTCATGCTCCTGCACCACCACAAAATGCCTCCACATCACCTTCTTATAATATTACTTCTAAGCATGAAGCAAACGATGATGATACGATCGAAAAGATCAAGCTTGGATTATCGAGAGCAATCATACACGAGCCACAGACAACCAACAAACCTTGGGACGCTCATGGAGCATCATCTGCAAACTTTCCTGGATTCCATTACGCCCTACCATCGTTTGGTCATGAAGGAGACGGATCTCCCTGGAGCCAGAGCATGTGCACTGGAAGCACATGCACCGCAGGTGACCAGCAACCGCAGTTTCACTCAAAAATGCCCGATAGAAACGCCGATGACTCTGAAGAAGAAAAAGATACAAGACATGTTTCAAGTATCTTCAGCCCCGACTGTGACTTGTGGGATCTACCTTCCAACGATCTCATGAATCCTTTAGTATAGACAAGATCTAAGATTGGA"

sgDNA:
GGAAGAGCTGTAGGTTACGCTGG

[Colelyman]

Hi @Gabelberg,

Thanks for using CRISPResso and sorry that you are running into this error. From what I can initially tell, this doesn't seem to be your fault. Would you mind providing the following information?

• What version of CRISPResso are you using?
• What are the original CRISPRessoBatch parameters (along with the batch parameter file if available)?
• What are the parameters for the separate CRISPResso that gives you the error above?

Thanks for reporting this, and we want to get to the bottom of it!

Thanks,
Cole

[Gabelberg]

Hi Cole,
many thanks for your really rapid response!
I am using CRISPResso 2.3.3, installed with conda a couple of days ago.
Here are the original CISPRessoBatch parameters:
CRISPRessoBatch
--write_detailed_allele_table --bam_output --place_report_in_output_folder
--output_folder $WD/crispresso/try1
--verbosity 3 --n_processes 16
--batch_settings $WD/crispresso/batchfile

and then I tried a more stringent run, in case that could change the problem (it didn't) adding
--min_average_read_quality 20 --min_single_bp_quality 10 --min_bp_quality_or_N 20

These are the parameters suggested for the single run:
CRISPResso -o "/mnt/Memorione_1/Data/Cichorium/demultiplexing_reads/CRISPRessoBatch_on_batchfile" --name S113_10A --fastq_r1 /mnt/Memorione_1/Data/Cichorium/demultiplexing_reads/fwdcartelleconidemux/S113_cost/S113_10A_R1.fastq --fastp_command fastp --max_rows_alleles_around_cut_to_plot 50 --aln_seed_count 5 --flexiguide_gap_extend_penalty -2 --fastq_r2 /mnt/Memorione_1/Data/Cichorium/demultiplexing_reads/fwdcartelleconidemux/S113_cost/S113_10A_R2.fastq --place_report_in_output_folder --prime_editing_pegRNA_scaffold_min_match_length 1 --aln_seed_min 2 --exclude_bp_from_right 15 --prime_editing_gap_extend_penalty 0 --aln_seed_len 10 --min_single_bp_quality 0 --bam_output --flexiguide_homology 80 --plot_window_size 20 --min_paired_end_reads_overlap 10 --flash_command None --write_detailed_allele_table --amplicon_seq ATATAAAGAAAAGAAAAGGGGAGAGGATGGGTCGAATCCCATGTTGCGAAAAGGAGAGTGTGAAGAAGGGTCAGTGGACCCCTGAAGAAGATCATAAGCTGTCTTCCTACATCGCTCAACATGGGACTCGTAATTGGCGCCTCATTCCCAAAAATGCAGGCCTCCAAAGATGTGGGAAGAGCTGTAGGTTACGCTGGACTAACTACCTACGCCCTGATTTAAAACATGGACAGTTCTCCGATGCTGAAGAACAAATCATTGTCAGGCTACATTCCGTTCTAGGCAACAGGTATAAATAACACATAAAATGTATGCATATGTATGTATGTATGTGCATGTATGTGTGTGTGTATCCATTCTCATGACCAAACTCTGAGAGATTGTATCTATACAATGCAAGTACAGGTGGTCCGTGATAGCGGCACAGCTGCCAGGACGAACAGACAATGATGTGAAGAACCATTGGAACACGAAACTGAAAAAGAAGCTGTCAGGTATGGGAATCGATCCCGTTACGCATAAGCCTTACTCCCACCTCATGGCGGAGATAGCCACCACTCTCGCTCCTCCACAGGTGGCTAACCTCGCAGAAGCAACTCTGGGATGTTTGAAAGATGAGATGCTTCATCTTCTAACCAAGAAGCACATAGATATTCAATTCCAATCCCCTGCTCATGCTCCTGCACCACCACAAAATGCCTCCACATCACCTTCTTATAATATTACTTCTAAGCATGAAGCAAACGATGATGATACGATCGAAAAGATCAAGCTTGGATTATCGAGAGCAATCATACACGAGCCACAGACAACCAACAAACCTTGGGACGCTCATGGAGCATCATCTGCAAACTTTCCTGGATTCCATTACGCCCTACCATCGTTTGGTCATGAAGGAGACGGATCTCCCTGGAGCCAGAGCATGTGCACTGGAAGCACATGCACCGCAGGTGACCAGCAACCGCAGTTTCACTCAAAAATGCCCGATAGAAACGCCGATGACTCTGAAGAAGAAAAAGATACAAGACATGTTTCAAGTATCTTCAGCCCCGACTGTGACTTGTGGGATCTACCTTCCAACGATCTCATGAATCCTTTAGTATAGACAAGATCTAAGATTGGA --conversion_nuc_to T --trimmomatic_command None --default_min_aln_score 60 --flexiguide_gap_open_penalty -20 --quantification_window_center -3 --flexiguide_seq None --max_paired_end_reads_overlap None --needleman_wunsch_gap_open -20 --amplicon_name Reference --prime_editing_pegRNA_extension_quantification_window_size 5 --verbosity 4 --config_file None --guide_seq GGAAGAGCTGTAGGTTACGCTGG --min_bp_quality_or_N 0 --min_frequency_alleles_around_cut_to_plot 0.2 --exclude_bp_from_left 15 --quantification_window_size 1 --needleman_wunsch_gap_extend -2 --n_processes 1 --min_average_read_quality 0 --prime_editing_gap_open_penalty -50 --conversion_nuc_from C --needleman_wunsch_gap_incentive 1 --needleman_wunsch_aln_matrix_loc EDNAFULL

In the attached zip folder you will find the batchfile, the fastq files of a sample (S113_10A) if you want to run a test, the standard error of a run I generated with --verbosity 1 (there are two lines I didn't catch the first time, at the start of the error: "/home/giovanni/miniforge3/envs/crispresso2_env/lib/python3.12/site-packages/CRISPResso2/CRISPRessoBatchCORE.py:220: PerformanceWarning: DataFrame is highly fragmented. This is usually the result of calling frame.insert many times, which has poor performance. Consider joining all columns at once using pd.concat(axis=1) instead. To get a de-fragmented frame, use newframe = frame.copy()
batch_params[arg] = getattr(args, arg)"

On my side, I will check the fastq files, since they were demultiplexed by a colleague of mine and I didn't verified them.

I thank you immensely for the support
crispresso.zip

[Gabelberg]

Hi Cole,
we found the error! They sent me the wrong sequence of the amplicons, since that was the region of interest, but the amplified sequence was different.
Once inserted the correct amplicon, everything worked fine!
Sorry for the fuss, and thanks again for the assistance!

(btw, this was the correct sequence, if this experience could be useful for a change in the error message
ATGGGTCGAATCCCATGTTGCGAAAAGGAGAGTGTGAAGAAGGGTCAGTGGACCCCTGAAGAAGATCATAAGCTGTCTTCCTACATCGCTCAACATGGGACTCGTAATTGGCGCCTCATTCCCAAAAATGCAGGCCTCCAAAGATGTGGGAAGAGCTGTAGGTTACGCTGGACTAACTACCTACGCCCTGATTTAAAACATGGACAGTTCTCCGATGCTGAAGAACAAATCATTGTCAGGCTACATTCCGTTCTAGGCAACAGGTATAAATAACACATAAAATGTATGCATATGTATGTATGTATGTGCATGTATGTGTGTGTGTATCCATTCTCATGACCAAACTCTGAGAGATTGTATCTATACAATGCAAGTACAGGTGGTCCGTGATAGCGGCACAGCTGCCAGGACGAACAGACAATGATG )

[Colelyman]

Hi @Gabelberg,

Great to hear that you were able to get the correct sequence and that things are working now!

Thanks,
Cole