unsupported operand type(s) for +=: 'int' and 'NoneType' in 2024

[Ohmori-Insilico]

We are having this problem using the SAM file to identify the off-target with Py3.

(base) user@ohmori-lab-ubuntu:~/Desktop/Guideseq 2024/guideseq/guideseq$ guideseq.py identify \

--aligned Data/aligned/VEGFA1_Tsai.sam
--genome Homo_sapiens_GRCh38ssembly.fa
--outfolder "/home/user/Desktop/Guideseq 2024/guideseq/guideseq/output"
--target_sequence GGGTGGGGGGAGTTTGCTCCNGG
--description "VEGFA1_site1"
[04/02 07:46:48AM][INFO][guideseq] Identifying offtarget sites...
[04/02 07:46:48AM][INFO][identifyOfftargetSites] Processing SAM file /home/user/Desktop/Guideseq 2024/guideseq/guideseq/output/aligned/VEGFA1_Tsai.sam
[04/02 07:47:01AM][ERROR][guideseq] Error identifying offtarget sites.
[04/02 07:47:01AM][ERROR][guideseq] Traceback (most recent call last):
File "/home/user/miniconda3/bin/guideseq.py", line 239, in identifyOfftargetSites
identifyOfftargetSites.analyze(samfile, self.reference_genome, self.identified[sample], annotations,
File "/home/user/miniconda3/bin/identifyOfftargetSites.py", line 314, in analyze
chromosome_position.addPositionBarcode(chromosome, read_position, strand, barcode, primer, count)
File "/home/user/miniconda3/bin/identifyOfftargetSites.py", line 60, in addPositionBarcode
self.chromosome_barcode_dict[chromosome][position][strand][barcode] += count
TypeError: unsupported operand type(s) for +=: 'int' and 'NoneType'

Samfile size: 7GB.
I used UMI, consolidation and aligned data. But problem is still persisted.
Test with EMX1 data from Guideseq insruction, it is working.
Is file size matter ?

Is there any solution to complete this task ?

Thanks a lot.

Neme.