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I am trying to perform a cellranger count on fastq files generated from a 10x genomics single cell RNA Seq run.

Just to provide some background, I ssh’d into the AWS using our AWS IP with MobaXTerm linux command line.

My files are in the Isilon. The file folders are path/to/runs/ - this folder contains the .fastq files for each sample (ex: SI_GA_A1_S1, SI_GA_B1_S2, SI_GA_C1_S3, and SI_GA_D1_S4). I can see these folders listed in the linux command line.

The cellranger is found when I write the following:

path/to/cellranger-3.0.1/cellranger-3.0.1

then ls brings up 13 items located in the folder, one of them being cellranger.

When I write the following command,

/path/to/cellranger-3.0.1/cellranger-3.0.1/cellranger count \
    --id=run2\
    --transcriptome=/path/to/refseq/refdata-cellranger-hg19-3.0.0\
    --sample=sample1\
    --expect-cells=5000

and it gives me:

-bash: /path/to/cellranger-3.0.1/cellranger-3.0.1/cellranger: No such file or directory

This looks correct from what the 10x website says for single cell RNA Seq. Please advise.

$ ls -l /path/to/cellranger-3.0.1/cellranger-3.0.1/cellranger 
-rwxrwx--- 1 1003080 1000000 460 Dec 5 19:41 /path/to/cellranger-3.0.1/cellranger-3.0.1/cellranger
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    $\begingroup$ You're using the wrong path. What does ls -l /path/to/cellranger-3.0.1/cellranger-3.0.1/cellranger return? Don't you want /path/to/cellranger-3.0.1//cellranger instead? $\endgroup$ – terdon May 28 at 14:46
  • $\begingroup$ @terdon, an ls -l /path/to/cellranger-3.0.1/cellranger-3.0.1/cellranger returns: -rwxrwx--- 1 1003080 1000000 460 Dec 5 19:41 /[insert isilon path here]/[insert windowsID here]/cellranger-3.0.1/cellranger-3.0.1/cellranger. why do you think it is the wrong path? if I do instead ls -l /path/to/cellranger-3.0.1//cellranger , it gives: ls: cannot access /[insert isilon path here]/[insert windowsID here]/cellranger-3.0.1/cellranger: No such file or directory $\endgroup$ – sv1234 May 28 at 15:02
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    $\begingroup$ Please edit your question to add clarifications. Comments are easy to miss, hard to read and can be deleted without warning. Use the formatting tools to format them as code so we can see what you are running. $\endgroup$ – terdon May 28 at 16:07
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    $\begingroup$ Please try and use consistent paths. This sort of issue is usually caused by a tiny detail, and it's hard to catch when you use both /path/to/ and /[insert isilon path here]/[insert windowsID here]. Also, the output of ls -l /path/to/cellranger-3.0.1/cellranger-3.0.1/cellranger seems to have an extra . at the end. That isn't actually in your output, right? $\endgroup$ – terdon May 28 at 16:31
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    $\begingroup$ Something is wrong here. The error tells you that /path/to/cellranger-3.0.1/cellranger-3.0.1/cellranger doesn't exist, but ls /path/to/cellranger-3.0.1/cellranger-3.0.1/cellranger shows the file. Are you 100% sure the paths you are using in your question are correct? Could you show us the real paths used? Are there any spaces in any of the paths? Any capital letters that you've changed to lowercase? $\endgroup$ – terdon May 28 at 18:41
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this has been resolved. I downloaded the windows file and this caused trouble but when downloading the file within the Linux, it seems to be working.

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It could be that cellranger does not have execute permissions. Try

chmod u+x [insert internal isilon path here]/[insert windows username here]/cellranger-3.0.1/cellranger-3.0.1/cellranger

(or the Windows equivalent, which seems to be to give Execute permission using the GUI)

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  • $\begingroup$ I tried this as you wrote and it says permission denied in linux command line. what is the windows equivalent? can I do this permission sharing in the Windows? $\endgroup$ – sv1234 May 28 at 14:43
  • $\begingroup$ This isn't a permissions issue, the error is about a wrong path. If the file existed but didn't have execute permissions, you would get a Permission denied error instead. $\endgroup$ – terdon May 28 at 14:45
  • $\begingroup$ @terdon, I get a Permissions denied error $\endgroup$ – sv1234 May 28 at 15:05
  • $\begingroup$ From your update, you seem to have the requisite permissions. @terdon Before OP's update, it could have been a permissions issue, and I wanted to ensure that was not the case. OP seems to be on a Windows machine, IDK how permissions work on Windows. $\endgroup$ – Ram RS May 28 at 17:35
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    $\begingroup$ The OP is connecting to an AWS instance running Linux (according to the question, anyway) and the error messages on Linux are quite specific. If the file exists but isn't executable, you would never get a file not found error. $\endgroup$ – terdon May 28 at 17:40

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