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Issue Running assign_gRNA_cells.py #3

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KASHISH72 opened this issue Jan 2, 2018 · 17 comments
Closed

Issue Running assign_gRNA_cells.py #3

KASHISH72 opened this issue Jan 2, 2018 · 17 comments

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@KASHISH72
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Hey
I am trying to run the whole pipeline for crop-seq, i am at the assign_gRNA_cells.py step and it is giving error.
File "assign_gRNA_cells.py", line 359, in
prj.add_sample_sheet()
File "crop-seq-final_version/src/looper/looper/models.py", line 546, in getattr
AttributeError: add_sample_sheet

Please let me know if you have any idea about the error

Thanks,
Kashish Chetal
@afrendeiro
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Hi, and thanks for the interest.
This project was run with an older version of looper (prior to the 0.4 release). Since then add_sample_sheet is called by the __init__ method when initializing a new Project. You can therefore simply skip that line if you use for example looper >=0.6.

@KASHISH72
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Hello
Thanks for the quick response but it is still giving the error.

Traceback (most recent call last):
File "src/assign_gRNA_cells.py", line 359, in
prj.paths.results_dir = results_dir = os.path.join("results")
File "crop-seq-final_version/src/looper/looper/models.py", line 546, in getattr
AttributeError: paths

@afrendeiro
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afrendeiro commented Jan 2, 2018 •
edited
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That is a different error. That line to is also not compatible with newer looper versions. Those variables are in fact not used in the entire script, so please also skip it.

@KASHISH72
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Now it is giving a different error

Traceback (most recent call last):
File "src/assign_gRNA_cells.py", line 373, in
reads = get_reads_in_construct(bam, sel_guide_annotation)
File "src/assign_gRNA_cells.py", line 37, in get_reads_in_construct
chrom_size = len(prj.config['crop-seq']['u6'] + guide_seq + prj.config['crop-seq']['rest'])
File "crop-seq-final_version/src/looper/looper/models.py", line 546, in getattr
AttributeError: config

@afrendeiro
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Alright, sorry. In newer looper versions the config attribute was merged into the Project object directly.
Please see commit 7b27527 with these instances updated.

@KASHISH72
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KASHISH72 commented Jan 2, 2018 via email

@nsheff
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nsheff commented Jan 2, 2018

Sorry for these annoyances. This stuff is stabilizing, though, so these should become less of an issue going forward :)

@afrendeiro
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Hi Kashish, I'm not sure about what is happening. Is the error message complete?
I can't debug right now since I do not have access to the files, will have to have a look next week.

@KASHISH72
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KASHISH72 commented Jan 2, 2018 via email

@afrendeiro
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Ah, okay. It seems that your BAM file does not contain "DNMT3B_1_chrom" as a reference (chromosome). Did you map your reads to a reference containing the artificial gRNA chromosomes as described here?
To confirm, see if these references are in the header of the BAM file.

@KASHISH72
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KASHISH72 commented Jan 2, 2018 via email

@afrendeiro
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So "DNMT3B_1_chrom" is not present in the refence genome or in the BAM file?
I assume you also ran that command to process the data with the Drop-seq pipeline or else you wouldn't have the mapped BAM files yet. Are you trying to run the same experiments as published or some new experiments? If the same, the mapped BAM files should definitely contain reads in all of the gRNA chromosomes. Please check the pipeline log files for errors or warnings that show that the pipeline completed successfully and there aren't for example truncated files for some reason.

@KASHISH72
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KASHISH72 commented Jan 2, 2018 via email

@afrendeiro
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Hmm that's odd then (sorry attachments don't work in the github issue tracker). Is it only this particular gRNA chromosome missing or the others too? For that experiment in particular there should be reads from 4 different gRNAs (annotated here) I believe.

@KASHISH72
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KASHISH72 commented Jan 2, 2018 via email

@yysheep2020
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Hi, I would like to ask when I will use "cellranger count" function, did you make the ref genome by yourself. More specifically, do we need to add sgRNA sequence into the human reference genome?

@afrendeiro
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Hi @yysheep2020, for the CROP-seq paper we didn't use neither 10X data or cellranger.
You can however run cellranger on CROP-seq data produced with the 10X platform by making a genome reference that includes gRNA sequences for example.

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4 participants
@afrendeiro @nsheff @KASHISH72 @yysheep2020