Issue with pathToMacs2

[spietrzak]

I have been working through the steps in the ArchR manual, and I have run into a problem with the Peak Calling using Macs2. I didn't have Macs2 already installed so I used Python3 to install it to my computer (Mac OS). However, when I try to either use the findMacs2 command or specify the path in the addReproduciblePeakSet command, I encounter errors.

For findMacs2(), I see the following output:
> pathToMacs2 <- findMacs2() Searching For MACS2.. Not Found in $PATH Not Found with pip Error in .checkPath(path2Bin, throwError = error) : object 'error' not found

For the addReproduciblePeakSet command, I see this output:
`> proj3 <- addReproduciblePeakSet(

• ArchRProj = proj3,
• groupBy = "Clusters",
• pathToMacs2 = "~/MyPythonEnv/bin"
• )
  Error in system2(path, "callpeak -h", stdout = TRUE, stderr = TRUE) :
  error in running command`

This was after updating to a more recent version of ArchR (0.9.5 from 0.9.3). Previously, the second command produced an error saying that the path that I had specified wasn't in my $PATH, even though I had added it to this. These new attempts after updating ArchR didn't produce any log files, but I have attached those from before the update for you. I am also attaching a screenshot of the folders in my $PATH which shows that the folder ~/MyPythonEnv/bin, which contains the Macs2 executable is, is a part of my $PATH.

Any advice would be greatly appreciated. Let me know if any additional information would be helpful.

ArchR-addReproduciblePeakSet-699c7288aeb7-Date-2020-06-08_Time-16-04-38.log
ArchR-addReproduciblePeakSet-699c7288aeb7-Date-2020-06-08_Time-16-04-38.log

[rcorces]

The parameter pathToMacs2 asks for the "full path to the MACS2 executable". You are both providing an abbreviated path (using "~") and the path to the folder containing MACS2 rather than the full path to the actual executable file. If you provide the full path all the way to the executable file it should work.

[spietrzak]

Thanks
ArchR-addReproduciblePeakSet-6eae20692928-Date-2020-06-08_Time-16-49-10.log
Ryan.

I changed to the full path and it looks like it was initially running normally, but I encountered a different error. In the output, there were several lines where it was saying "No such file or directory" followed by several error messages:

`

proj3 <- addReproduciblePeakSet(

• ArchRProj = proj3,
• groupBy = "Clusters",
• pathToMacs2 = "/Users/stefanpietrzak/MyPythonEnv/bin/macs2"
• )
  ArchR logging to : ArchRLogs/ArchR-addReproduciblePeakSet-6eae20692928-Date-2020-06-08_Time-16-49-10.log
  If there is an issue, please report to github with logFile!
  2020-06-08 16:49:11 : Peak Calling Parameters!, 0.006 mins elapsed.
  Group nCells nCellsUsed nReplicates nMin nMax maxPeaks
  C1 C1 65 60 2 40 40 30000
  C2 C2 193 193 2 40 153 96500
  C3 C3 1946 540 2 40 500 150000
  C4 C4 2466 664 4 45 500 150000
  C5 C5 1342 1286 5 73 480 150000
  C6 C6 212 212 3 48 91 106000
  C7 C7 4104 1581 5 64 500 150000
  C8 C8 4921 1840 5 75 500 150000
  C9 C9 551 527 3 44 292 150000
  C10 C10 5434 1000 2 500 500 150000
  C11 C11 2474 1000 2 500 500 150000
  C12 C12 3216 847 2 347 500 150000
  C13 C13 515 501 2 161 340 150000
  C14 C14 490 477 2 209 268 150000
  C15 C15 1091 1041 5 65 456 150000
  C16 C16 366 320 5 55 78 150000
  C17 C17 2814 601 3 46 500 150000
  C18 C18 5672 1636 5 53 500 150000
  C19 C19 2995 1179 4 42 500 150000
  C20 C20 3869 2023 5 319 500 150000
  C21 C21 46 42 2 26 28 21000
  2020-06-08 16:49:11 : Batching Peak Calls!, 0.006 mins elapsed.
  2020-06-08 16:49:11 : Batch Execution w/ safelapply!, 0 mins elapsed.
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  2020-06-08 16:51:36 : Identifying Reproducible Peaks!, 2.428 mins elapsed.
  Error in .safelapply(seq_along(outSummitList), function(i) { :
  Error Found Iteration 1 :
  [1] "Error in .logError(e, fn = ".identifyReproduciblePeaks", info = prefix, : \n Exiting See Error Above\n"
  <simpleError in .logError(e, fn = ".identifyReproduciblePeaks", info = prefix, errorList = errorList, logFile = logFile): Exiting See Error Above>
  Error Found Iteration 2 :
  [1] "Error in .logError(e, fn = ".identifyReproduciblePeaks", info = prefix, : \n Exiting See Error Above\n"
  <simpleError in .logError(e, fn = ".identifyReproduciblePeaks", info = prefix, errorList = errorList, logFile = logFile): Exiting See Error Above>
  Error Found Iteration 3 :
  [1] "Error in .logError(e, fn = ".identifyReproduciblePeaks", info = prefix, : \n Exiting See Error Above\n"
  <simpleError in .logError(e, fn = ".identifyReproduciblePeaks", info = prefix, errorList = errorList, logFile = logFile): Exiting See Error Above>
  Error Found Iteration 4 :
  [1] "Error in .logError(e, fn = ".identifyReproduciblePeaks", info = prefix, :
  In addition: Warning message:
  In mclapply(..., mc.cores = threads, mc.preschedule = preschedule) :;
  21 function calls resulted in an error
  `
  I'm not sure what the issue could be here. I have attached the associated log file for this

[spietrzak]

One thing I noticed in the log file is that it appears that for each cluster, it is trying to use an insertions.bed file in the "Outputs/PeakCalls/InsertionBeds/" folder, which is empty when I went into that folder. Were these supposed to be created in a previous step that I may have missed?

[rcorces]

Correct. I will update the documentation to make this more explicit. Peak calling is performed on group coverages which are created using addGroupCoverages(). Strongly recommend going through the full tutorial because these steps can be quite complicated.
https://www.archrproject.com/bookdown/making-pseudo-bulk-replicates.html

[spietrzak]

Thanks for your quick responses. I have performed the addGroupCoverages step, and it didn't produce any errors, yet I still don't see anything in the InsertionBeds folder. I tried again, but still no luck. The only step that I haven't performed is the integration with scRNA-seq in section 8 of the manual, but I didn't think that would effect these steps. I have attached the log file for this and a screenshot of the output in R. Do you have any thoughts on this? Thanks again for your all of your help.
ArchR-addGroupCoverages-6eae580d11b7-Date-2020-06-08_Time-20-19-42.log

[rcorces]

Sorry - I read your previous comment incorrectly. While addGroupCoverages() is a prerequisite for peak calling, it has nothing to do with the InsertionBeds folder.
The problem you are having is that one of your directories "Single Cell Project" has spaces in the name. We have encountered this error in other parts of ArchR. If it isnt too much of a hassle, I would re-run everything from a directory path that has no spaces. If that is too inconvenient, you could try saving your ArchRProject using saveArchRProject() to a new directory path that doesnt contain spaces but I'm not 100% sure how that will go.

@jgranja24 - flagging this as another "spaces in file/directory names" problem

[spietrzak]

Thanks.

I changed the name of the folder so that there are no spaces and tried moving the folder for the R-project to a different directory, but in each case, it says in the log file that the outputDirectory still has the original path with the folder with spaces in it. Do you know if there is a way to easily change this in the ArchR project object without starting over and making a new ArchRProject?

[rcorces]

The outputDirectory is stored in proj@projectMetadata$outputDirectory. You should be able to change the value of this:

proj@projectMetadata$outputDirectory <- "/New/Output/Directory"

you can validate that this worked by doing getOutputDirectory(proj)

[sylestiel]

@rcorces
Unfortunately I have encountered the same error:
Error in system2(path, "callpeak -h", stdout = TRUE, stderr = TRUE) :
error in running command

I have run the addReproduciblePeakSet command numerous times without any issue. Hence, I'm confounded. The log file is attached. Hope you can help me troubleshoot.
ArchR-addGroupCoverages-8e7d106fa905-Date-2022-06-21_Time-12-38-45.log