Fixing ScATACpipe: Permission Denied Error

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Fixing "Permission Denied" Errors in scATACpipe Test Data

Introduction: Decoding the "Permission Denied" Mystery

Hey there, bioinformatics enthusiasts! Have you ever hit a wall when trying to run a pipeline, staring at a frustrating "Permission denied" error? It's a common hurdle, especially when working with shared computing environments like SLURM clusters. This article digs deep into a specific issue encountered while running the scATACpipe test data. The user, hukai916, ran into a roadblock, and we'll break down the problem, the troubleshooting steps, and hopefully, provide a solution to get your scATACpipe runs back on track. This article will help you understand the root cause of the error. We will also help you fix the error by walking through the troubleshooting steps. The error typically surfaces when the pipeline is trying to access files, but something's blocking it. Let's get into the nitty-gritty of the error, the environment, and the steps to get things working smoothly. This particular error message, as seen in the scATACpipe test data, pops up when the pipeline attempts to access the FASTQ files. To fully grasp the situation, it's essential to understand the components involved: the SLURM cluster, the Nextflow pipeline manager, the scATACpipe itself, and the file permissions on the shared storage. The core of the problem lies in the interaction between these elements. Let's unravel this mystery together! The user's goal is to successfully run the scATACpipe test data on a SLURM cluster and understand why it failed.

Understanding the Error

The primary symptom is the "Permission denied" message, specifically occurring during the ADD_BARCODE_TO_READS step. The error message indicates that the pipeline cannot access the FASTQ files, despite the files seemingly having the correct read permissions. The full error message looks something like this:

ERROR ~ Error executing process > 'SCATACPIPE:PREPROCESS_DEFAULT:ADD_BARCODE_TO_READS (1)'

Caused by:
  Process `SCATACPIPE:PREPROCESS_DEFAULT:ADD_BARCODE_TO_READS (1)` terminated with an error exit status (126)

Command executed:

  # use the first read length from fastq file to determine the length since -b is required by sinto.
  barcode_length=$((zcat downsample_10p_atac_pbmc_500_nextgem_S1_L002_R2_001.fastq.gz || true) | awk 'NR==2 {print length($0); exit}')
  # sinto barcode  --barcode_fastq downsample_10p_atac_pbmc_500_nextgem_S1_L002_R2_001.fastq.gz --read1 downsample_10p_atac_pbmc_500_nextgem_S1_L002_R1_001.fastq.gz --read2 downsample_10p_atac_pbmc_500_nextgem_S1_L002_R3_001.fastq.gz -b $barcode_length
  addbarcodes_parallel.py downsample_10p_atac_pbmc_500_nextgem_S1_L002_R2_001.fastq.gz $barcode_length downsample_10p_atac_pbmc_500_nextgem_S1_L002_R1_001.fastq.gz downsample_10p_atac_pbmc_500_nextgem_S1_L002_R3_001.fastq.gz 12
  
  # remove sequence description in + line, otherwise, cutadapt may complain if it does not match with line 1:
  zcat downsample_10p_atac_pbmc_500_nextgem_S1_L002_R1_001.barcoded.fastq.gz | awk '{if ($1 ~/^
  zcat downsample_10p_atac_pbmc_500_nextgem_S1_L002_R3_001.barcoded.fastq.gz | awk '{if ($1 ~/^
  mv tem_downsample_10p_atac_pbmc_500_nextgem_S1_L002_R1_001.barcoded.fastq.gz downsample_10p_atac_pbmc_500_nextgem_S1_L002_R1_001.barcoded.fastq.gz
  mv tem_downsample_10p_atac_pbmc_500_nextgem_S1_L002_R3_001.barcoded.fastq.gz downsample_10p_atac_pbmc_500_nextgem_S1_L002_R3_001.barcoded.fastq.gz
  
  # rename the files:
  mv downsample_10p_atac_pbmc_500_nextgem_S1_L002_R1_001.barcoded.fastq.gz R1_pbmc_500_10p_1.barcoded.fastq.gz
  mv downsample_10p_atac_pbmc_500_nextgem_S1_L002_R3_001.barcoded.fastq.gz R2_pbmc_500_10p_1.barcoded.fastq.gz
  mv downsample_10p_atac_pbmc_500_nextgem_S1_L002_R2_001.fastq.gz barcode_pbmc_500_10p_1.fastq.gz # rename input is fine to nextflow

Command exit status:
  126

Command output:
  (empty)

Command wrapper:
  /var/spool/slurm/d/job5373455/slurm_script: line 338: /ngsprojects/plantatac/data_archive/atacseq/pipelines/scATACpipe/test_data1/downsample_10p_atac_pbmc_500_nextgem_S1_L002_R1_001.fastq.gz: Permission denied

Work dir:
  /scratch/plantatac/scATACpipe/e1/f34476f34ad8c8c70f0180920bec8d

This output is crucial because it gives us a direct pointer to the problem. It states the specific file that the pipeline tried to access (downsample_10p_atac_pbmc_500_nextgem_S1_L002_R1_001.fastq.gz) and the resulting permission error. The error occurs when the script tries to execute the addbarcodes_parallel.py script. The script aims to add barcodes to reads, which is a critical step in processing scATAC-seq data. The error itself, "Permission denied," means the user running the pipeline (the SLURM job) does not have the necessary permissions to read this particular file. This is happening despite the apparent read permissions on the FASTQ files. The files themselves have -rw-r--r-- permissions, meaning the owner has read and write permissions, and everyone else has read permissions. The key to resolving this kind of issue lies in pinpointing the exact user account under which the SLURM job is running and confirming its access to the relevant files. Understanding the exact user context is crucial in the SLURM environment because it manages the allocation of resources and user identities. The next step is to examine the file paths and ensure the SLURM job can access them.

Reproducing the Error: Steps to Recreate the Issue

To ensure we're all on the same page, here are the exact steps hukai916 used to encounter the "Permission denied" error. Knowing the precise method of reproduction helps us narrow down the potential causes. Remember, the goal is to make the error occur consistently so that we can verify our fix. Here’s how the user set up the test:

  1. Cloning scATACpipe: The first step involves getting the scATACpipe code from GitHub. Make sure you have the latest version to ensure you're working with the most up-to-date code. This is a crucial first step, as any changes in the code can affect the pipeline's behavior and the location of the files that it accesses. The user is advised to clone the scATACpipe repository from GitHub to get started.

  2. Running with Test Data: The core of the problem lies in running the pipeline with the provided test data and the command line provided in the documentation. The user executed the command: nextflow run main.nf -c scatac-seq-slurm.config -profile singularity --preprocess default --outdir res_test_data1 --input_fastq assets/sample_sheet_test_data1.csv --ref_fasta_ensembl homo_sapiens --species_latin_name 'homo sapiens'. Pay close attention to this command. It specifies the configuration file (scatac-seq-slurm.config), the profile (singularity), the preprocessing steps, the output directory (res_test_data1), the input file (assets/sample_sheet_test_data1.csv), and the reference genome.

  3. Configuration: The configuration file is essential for adapting the pipeline to the SLURM environment. The scatac-seq-slurm.config file sets the workDir and tells Nextflow how to execute the jobs using SLURM. It also specifies the executor as 'slurm'. The workDir is set to /scratch/plantatac/scATACpipe. Make sure the working directory has sufficient space and access permissions. If the working directory isn't set up correctly, the pipeline will encounter errors because it cannot write intermediate files. The configuration file scatac-seq-slurm.config is crucial.

    workDir = '/scratch/plantatac/scATACpipe'

executor {
    name = 'slurm'
}

  4. Error Encountered: After running the pipeline, the user observed the "Permission denied" error, as described earlier. If the command and the configuration are set up correctly, you should be able to reproduce the error by running the same steps. This ensures that the steps are replicable.

Troubleshooting: Root Cause Analysis and Potential Fixes

Alright, let's roll up our sleeves and troubleshoot this "Permission denied" error. Here's a breakdown of the diagnostics and potential fixes. Remember, the key is to methodically eliminate possible causes. The core of the problem often lies in file permissions or the user context within the SLURM environment. Let's dig in!

Verify File Permissions and Ownership

  1. Check Permissions: The FASTQ files themselves have -rw-r--r-- permissions. This means the owner (in this case, iabos) has read and write permissions, and everyone else (group and others) has read permissions. The files seem to be readable. Verify that the files have the correct permissions, and that the SLURM job's user can read these files. Use ls -l on the files to see their permissions, owner, and group.
  2. User Context in SLURM: This is crucial. When a job runs on a SLURM cluster, it runs under a specific user account. The most common pitfall is that the user running the Nextflow pipeline may not be the same user that owns the FASTQ files or has access to the directory. Identify the actual user account under which the SLURM job is running. This is usually the same user you use to log into the cluster. This is where the issue usually lies. Use whoami to check your current user. In a SLURM job, this might be a different user than the one you are logged in as. To check the job's user, you can add whoami at the beginning of your Nextflow script.
  3. Group Membership: Ensure that the user running the SLURM job is part of a group that has access to the FASTQ files. The files are owned by the plantatac group. The user running the pipeline must be a member of the plantatac group. The group affiliation is essential for access.
  4. File Paths: Double-check the file paths in your sample_sheet_test_data1.csv and the Nextflow configuration to make sure they are correct. Even a small typo can lead to a "Permission denied" error. Validate the paths of the FASTQ files. Incorrect paths are a common cause of this error. Review the paths in the sample_sheet_test_data1.csv file, making sure they are correct.
  5. NFS Mounts: NFS (Network File System) mounts are common in HPC environments. Sometimes, there can be issues with how the NFS mount is configured, leading to permission problems. Make sure the NFS mount (/ngsprojects/plantatac/...) is correctly configured and that the user running the pipeline has the appropriate access rights to that mount. Check that the NFS mount has proper read access for the user.

Common Solutions and Workarounds

  1. Correct User Context: The most common fix is to ensure that the user running the SLURM job has the correct permissions. Make sure the SLURM job runs under the user who owns the files or a user who is part of the group that owns the files. You might need to specify the user in your SLURM submission script using the #SBATCH -u flag. Modify the SLURM job to use the correct user.
  2. Group Membership: Verify that the user is part of the correct group using the groups command. If not, you might need to request group membership from your system administrator. If the user is not part of the correct group, request access.
  3. Check the Working Directory: Ensure that the working directory specified in the Nextflow configuration (/scratch/plantatac/scATACpipe) has the correct permissions. The user running the pipeline needs write access to this directory. If the permissions on the working directory are incorrect, the pipeline may not be able to write intermediate files, leading to errors. The user needs to have write permissions in the working directory.
  4. Use Absolute Paths: In your Nextflow script and sample sheet, use absolute paths instead of relative paths. This helps prevent confusion about the location of the files. The absolute path eliminates ambiguity in the file location.
  5. Singularity/Docker: Make sure your container setup (Singularity in this case) has the correct permissions to access the files. In some cases, the container might be running as a different user, which could cause permission problems. If you're using Singularity (as in this case), make sure the container is configured to map the host user ID to the user ID inside the container. This is essential for ensuring that the containerized processes can access files owned by the host user. Ensure that the container has the correct permissions to access the files.
  6. Contact System Administrator: If you've tried everything and still face issues, it's time to reach out to your system administrator. They can help diagnose more complex permission problems or issues with the SLURM configuration or the NFS mounts. Get help from your system administrator if needed.

Advanced Troubleshooting: Digging Deeper

If the basic checks don't work, here are some advanced steps to try. These involve more detailed diagnostics and can help pinpoint more subtle permission problems. Let's get more specific and explore these advanced troubleshooting methods.

Debugging within Nextflow

  1. Add Debug Statements: You can add debug statements to your Nextflow script. For example, insert touch /scratch/plantatac/scATACpipe/test.txt before the step where the error occurs. This can help you verify if Nextflow can even write to the working directory. Debugging within Nextflow can provide essential information.
  2. Inspect the Command: Examine the exact command that Nextflow is trying to execute. You can often see this in the Nextflow logs. Make sure that the command is what you expect it to be. This is crucial for understanding how the commands are executed.
  3. Run Locally: Try running the pipeline locally on your machine, outside the SLURM environment, to see if the error persists. This will help you isolate whether the problem is specific to the SLURM setup. Try running the pipeline outside the SLURM environment to isolate the issue.

SLURM-Specific Checks

  1. SLURM Job Script: Carefully review the SLURM job script generated by Nextflow or the one you are submitting. Make sure it specifies the correct user, group, and any necessary environment variables. The SLURM job script is key.
  2. SLURM Environment Variables: Check the SLURM environment variables within your job. These variables can sometimes affect file access. Verify that environment variables are set correctly within the SLURM job.

Addressing the "Skipping Files" Error with Nextflow 24.10.4

When upgrading to Nextflow 24.10.4, a new error surfaces: "Skipping files." This suggests a different issue related to file existence or accessibility. The output indicates that the FASTQC process is skipping files because they don't exist or can't be read. This is a crucial clue that points to problems in file paths or file access within your configuration. Here's a breakdown of the diagnostics and potential fixes. Understanding the new error is essential for a complete solution.

  1. File Path Verification: Double-check the file paths used by FastQC. Ensure that the files downsample_5p_atac_pbmc_500_nextgem_S1_L002_R1_001.fastq.gz, downsample_5p_atac_pbmc_500_nextgem_S1_L002_R3_001.fastq.gz, and downsample_5p_atac_pbmc_500_nextgem_S1_L002_R2_001.fastq.gz are located where FastQC expects them. Typos or incorrect paths are the most common culprits. Validate the file paths.
  2. Working Directory Context: The error message indicates that the working directory is /scratch/plantatac/scATACpipe/c7/bca77ce2eea2508d1d4fb6e1480e21. Confirm that the FASTQ files are accessible from within this working directory. Check the working directory context.
  3. Container Context: When using Singularity, the container's view of the file system might differ from the host's view. Ensure that the file paths inside the container match the paths on the host system. The container's view of the filesystem can be different.
  4. Input File Integrity: Verify that the input files are not corrupted or incomplete. Download and re-check the files to ensure that they are readable. Check the input file integrity.

Conclusion: Your Path to a Smooth scATACpipe Run

Navigating "Permission denied" errors and related issues in bioinformatics can be a challenge. We've explored the user's specific problem with scATACpipe, providing a step-by-step guide to troubleshooting. Remember, the key is methodical investigation: verify file permissions, double-check file paths, and understand the user context within your SLURM environment. The tips provided will help you overcome the challenges. By systematically working through these steps, you should be able to get your scATACpipe pipeline running smoothly, allowing you to focus on the science rather than the software. Good luck, and happy analyzing! Remember to always consult with your system administrator if you are still facing any problems.