ERROR: Offset around cut would be greater than reference sequence length.

[albustruong]

Describe the bug
CRISPResso version 2.2.14
[Command used]:
/home/albustruong/miniconda3/envs/ngs/bin/CRISPResso -r1 2404-1-A4.R1.clean.fastq -r2 2404-1-A4.R2.clean.fastq --amplicon_seq ATTGCAGAGAGGCGTATCATTTCGCGGATGTTCCAATCAGTACGCAGAGAGTCGCCGTCTCCAAGGTGAAAGCGGAAGTAGGGCCTTCGCGCACCTCATGGAATCCCTTCTGCAGCACCTGGATCGCTTTTCCGAGCTTCTGGCGGTCTCAAGCACTACCTACGTCAGCACCTGGGACCCCGCCACCGTGCGCCGGGCCTTGCAGTGGGCGCGCTACCTGCGCCACATCCATCGGCGCTTTGGTCGGCATGGCCCCAT --prime_editing_pegRNA_spacer_seq GGAATCCCTTCTGCAGCACC --prime_editing_pegRNA_extension_seq CCTTTTCGCTAGGTCCTCGACGTCTTCCCTA --exclude_bp_from_right 0 --exclude_bp_from_left 0 -wc -3 --plot_window_size 10 -n Quinn -o ./

[Execution log]:
Computing quantification windows
Traceback (most recent call last):
File "/home/albustruong/miniconda3/envs/ngs/lib/python3.10/site-packages/CRISPResso2/CRISPRessoCORE.py", line 1688, in main
this_exclude_idxs) = CRISPRessoShared.get_amplicon_info_for_guides(this_seq, this_guides, this_guide_mismatches, this_guide_names, this_guide_qw_centers,
File "/home/albustruong/miniconda3/envs/ngs/lib/python3.10/site-packages/CRISPResso2/CRISPRessoShared.py", line 1504, in get_amplicon_info_for_guides
raise BadParameterException(
CRISPResso2.CRISPRessoShared.BadParameterException: Offset around cut would be greater than reference sequence length. Please decrease plot_window_size parameter. Cut point: 251 window: 10 reference: 258

Parameter error, please check your input.

ERROR: Offset around cut would be greater than reference sequence length. Please decrease plot_window_size parameter. Cut point: 251 window: 10 reference: 258

Question 1
Why was the cut point is defaultly set at 251 when the reference length is only 258?

Decrease plot_window_size parameter as advised led to bug 2
CRISPResso version 2.2.14
[Command used]:
/home/albustruong/miniconda3/envs/ngs/bin/CRISPResso -r1 2404-1-A4.R1.clean.fastq -r2 2404-1-A4.R2.clean.fastq --amplicon_seq ATTGCAGAGAGGCGTATCATTTCGCGGATGTTCCAATCAGTACGCAGAGAGTCGCCGTCTCCAAGGTGAAAGCGGAAGTAGGGCCTTCGCGCACCTCATGGAATCCCTTCTGCAGCACCTGGATCGCTTTTCCGAGCTTCTGGCGGTCTCAAGCACTACCTACGTCAGCACCTGGGACCCCGCCACCGTGCGCCGGGCCTTGCAGTGGGCGCGCTACCTGCGCCACATCCATCGGCGCTTTGGTCGGCATGGCCCCAT --prime_editing_pegRNA_spacer_seq GGAATCCCTTCTGCAGCACC --prime_editing_pegRNA_extension_seq CCTTTTCGCTAGGTCCTCGACGTCTTCCCTA --exclude_bp_from_right 0 --exclude_bp_from_left 0 -wc -3 --plot_window_size 5 -n Quinn -o ./

[Execution log]:
Computing quantification windows
Traceback (most recent call last):
File "/home/albustruong/miniconda3/envs/ngs/lib/python3.10/site-packages/CRISPResso2/CRISPRessoCORE.py", line 1675, in main
pe_guides, pe_orig_guide_seqs, pe_guide_mismatches, pe_guide_names, pe_guide_qw_centers, pe_guide_qw_sizes, pe_guide_plot_cut_points = get_prime_editing_guides(this_seq, this_name, amplicon_seq_arr[0],
File "/home/albustruong/miniconda3/envs/ngs/lib/python3.10/site-packages/CRISPResso2/CRISPRessoCORE.py", line 1533, in get_prime_editing_guides
nicking_center_this_amp_seq = rev_coords_r[nicking_center_ref0] - coords_r[r0_end_loc]
IndexError: list index out of range

Unexpected error, please check your input.

ERROR: list index out of range

Request

Please help me fix this. Or is this because of the raw data?

Albus

[Colelyman]

Hi Albus,

Thanks for using CRISPResso! Sorry that you are running into this issue. Could you run the second command with the --debug flag and provide the output?

Thanks,
Cole

[albustruong]

Hi Cole,

I have rerun. Please see the log:

Describe the bug

CRISPResso version 2.2.14
[Command used]:
/home/albustruong/miniconda3/envs/ngs/bin/CRISPResso -r1 2404-1-A4.R1.clean.fastq -r2 2404-1-A4.R2.clean.fastq --amplicon_seq ATTGCAGAGAGGCGTATCATTTCGCGGATGTTCCAATCAGTACGCAGAGAGTCGCCGTCTCCAAGGTGAAAGCGGAAGTAGGGCCTTCGCGCACCTCATGGAATCCCTTCTGCAGCACCTGGATCGCTTTTCCGAGCTTCTGGCGGTCTCAAGCACTACCTACGTCAGCACCTGGGACCCCGCCACCGTGCGCCGGGCCTTGCAGTGGGCGCGCTACCTGCGCCACATCCATCGGCGCTTTGGTCGGCATGGCCCCAT --prime_editing_pegRNA_spacer_seq GGAATCCCTTCTGCAGCACC --prime_editing_pegRNA_extension_seq CCTTTTCGCTAGGTCCTCGACGTCTTCCCTA --exclude_bp_from_right 0 --exclude_bp_from_left 0 -wc -3 --plot_window_size 5 --debug -n albus -o ./

[Execution log]:
Alignment between extension sequence and reference sequence:
------------------------------------------------------------------------------TAGGG-------------------------------------------------------------------AAG--------ACGTCGAGGACCTAG---------------------------------------------------------------------CGAAAAGG------
ATTGCAGAGAGGCGTATCATTTCGCGGATGTTCCAATCAGTACGCAGAGAGTCGCCGTCTCCAAGGTGAAAGCGGAAGTAGGGCCTTCGCGCACCTCATGGAATCCCTTCTGCAGCACCTGGATCGCTTTTCCGAGCTTCTGGCGGTCTCAAGCACTACCTACGTC-AGCACCTGGGACCCCGCCACCGTGCGCCGGGCCTTGCAGTGGGCGCGCTACCTGCGCCACATCCATCGGCGCTTTGGTCGGCATGGCCCCAT
Computing quantification windows
Traceback (most recent call last):
File "/home/albustruong/miniconda3/envs/ngs/lib/python3.10/site-packages/CRISPResso2/CRISPRessoCORE.py", line 1675, in main
pe_guides, pe_orig_guide_seqs, pe_guide_mismatches, pe_guide_names, pe_guide_qw_centers, pe_guide_qw_sizes, pe_guide_plot_cut_points = get_prime_editing_guides(this_seq, this_name, amplicon_seq_arr[0],
File "/home/albustruong/miniconda3/envs/ngs/lib/python3.10/site-packages/CRISPResso2/CRISPRessoCORE.py", line 1533, in get_prime_editing_guides
nicking_center_this_amp_seq = rev_coords_r[nicking_center_ref0] - coords_r[r0_end_loc]
IndexError: list index out of range

Unexpected error, please check your input.

ERROR: list index out of range
CRISPResso_RUNNING_LOG.txt