LentiGuide library

[Jeremy Wen]

Hi Good morning

I have 2 forward reads files for both my control and treatment group. when I run only one forward reads from each sample, there were no error messages (at the end of email message)

But when I combined the 2 forwards and run the analysis, the error messages showed. and a lot of the plots are missing from the results of the analysis.

Can anyone let me know if anything wrong with my combined files and what I need to do the correct the error. 

Thank you very much

Jeremy

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Loading sgRNA read counts ...Computing fold-changes ...Computing p-values (Neg. Binomial model) ...Multiple tests correction ...Traceback (most recent call last):  File "RanksgRNAs.py", line 208, in <module>    PrepareGuideRanking(input1)   File "RanksgRNAs.py", line 139, in PrepareGuideRanking    multTest = multipletests(pval_0,alpha,padj)  File "/usr/local/lib/python2.7/dist-packages/statsmodels/stats/multitest.py", line 144, in multipletests    alphacSidak = 1 - np.power((1. - alphaf), 1./ntests)ZeroDivisionError: float division by zeroProcessing sample EarlyTreatment2_1 ... ++++++++++++++++++++++++++++++++++++++++++++++++

++++++++++++++++++++++++++++++++++++++++++++++++

Reading sgRNA read counts ...Traceback (most recent call last):  File "PlotCounts.py", line 218, in <module>    GOI_Scatterplot(input1)              File "PlotCounts.py", line 74, in GOI_Scatterplot    os.chdir(sgRNARanksDir)OSError: [Errno 2] No such file or directory: '/workingdir/Analysis/02_sgRNA-Ranking_Results/sgRNA_Rankings/'

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[Philipp N. Spahn]

Hi Jeremy,

can you share the link to your run?


Philipp

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-------------------------------
Philipp N. Spahn, PhD

Asst. Project Scientist
Lewis Laboratory
Biomedical Research Facility II
School of Medicine
University of California, San Diego
9500 Gilman Dr, Mail Code 0760
La Jolla, CA 92093-0760
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[Philipp N. Spahn]

Hi Jeremy,

it's difficult to diagnose from here without having your files, but my guess is that it must be a formatting issue from splitting and rejoining the files.

There is nothing wrong with keeping the files separate, even if they are from the same biological sample. You can simply treat them as technical replicates.

Also keep in mind that excessive read depth (beyond 50 million reads / sample) are typically not required for regular crispr screens and only add unnecessary computation time. If your read depth is beyond that, you can trim the files to reduce size.

I would recommend you talk your sequencing facility. They should be able to help you with preparing/trimming your files.

I hope this helps.

Best,
Philipp

On 12/15/2021 4:28 AM, Jeremy Wen wrote:

HI Philipp

the files I uploaded are not the same (files attached), they have different names and sizes. I guess the files are too big and they split into 2 when the core sent me the files. and when I upload the file, I combined the read files from the same sample into one file. Please let me know how I can resolve the error message. 

Thanks

Jeremy

On Mon, Dec 13, 2021 at 4:45 PM Philipp N. Spahn <psp...@eng.ucsd.edu> wrote:

Hi Jeremy,

can you remind me if this has been resolved.

It would cause an error if you accidentally uploaded the same reads file twice, especially for the control, or if these files happened to be identical (maybe due to a file naming error).

Philipp

On 12/6/2021 11:36 AM, Jeremy Wen wrote:

Hi Phillip 

Thanks for helping out. 

My failed run with combined files are here http://pinapl-py.ucsd.edu/run/1638531704_14593 

When I used only one of forward reads, it worked out, here is the link http://pinapl-py.ucsd.edu/run/1638371432_14531 

Thank you very much.

Best

Jeremy

[Jeremy Wen]

Hi Philipp

Thanks. It makes a lot sense to me. I will check with the bioinformatics people here. 

Best 

Jeremy