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🧪 New pipeline for detecting pseudouridine modification on RNA (BID-seq, etc)

Home Page: https://bidseq.chuan.science/

License: GNU General Public License v3.0

Dockerfile 3.20% Shell 8.91% Python 87.89%

rna pseudouridine modification single-base mrna rrna epitranscriptome epitranscriptomics

pseudou-bidseq's Issues

Detailed parameters of STAR

I'm sorry to bother you.
Can you provide your detailed parameters of STAR?
And I'm still confused about setting parameters of barcode. Because I have already cut the UMI sequence and barcodes. Do I still need to write "barcode: '-NNNNN'" in data.yaml?
Looking forward to your reply! I will really really really appreciate you!!
Best wishes

header of result

Hi @y9c , thanks very much!
i have got the final result. but i don't know what mean is the depth, gap,WT_ratio,WT_fraction. could you give me a detailed explanation ?
genome.zip

Error in rule gap_realign

Hi y9c!
I am trying your pseudoU-BIDseq pipeline using the SRRSRR15082607,SRR15082609 and SRR15082610.
And I have the following error when run the pipeline,

[Wed Jan 10 14:11:17 2024]
Error in rule gap_realign:
    jobid: 25
    output: .tmp/mapping_realigned_unsorted/HeLaWT-rep1-treated_run1_contamination.cram
    shell:
        
        /pipeline/bin/realignGap -r /disk/user_18/jky_project/genome/Mesomycoplasma_hyorhinis_ATCC_17981/Mesomycoplasma_hyorhinis_ATCC_17981.fna -i .tmp/mapping_unsort/HeLaWT-rep1-treated_run1_contamination.bam -o .tmp/mapping_realigned_unsorted/HeLaWT-rep1-treated_run1_contamination.cram
        
        (one of the commands exited with non-zero exit code; note that snakemake uses bash strict mode!)

Removing output files of failed job gap_realign since they might be corrupted:
.tmp/mapping_realigned_unsorted/HeLaWT-rep1-treated_run1_contamination.cram

I wonder what can I do, thank you so much!

barcode setting

Hi y9c!
I have a question here. I use the R2 reads for your pipeline，and there are 8nt UMI and 6nt random sequence in the 5' end, and i have already trimmed the 3' end adapter, how should i set the barcode, is "NNNNNNNNXXXXXX-" work? Looking forward to your reply, thanks!

Duplicated reads level is high

Hi, @y9c !
I have a question here. I use the BID-pipe for my data, and the duplication level is 40%. I also use 'seqkit rmdup' by the sequence to calculate the duplication level, it's only 20%. I want to know how the BID-pipe calculate the duplication level and what's the difference from the seqkit rmdup. Looking forward to your reply. Thanks a lot!

Filtering question

Hello, I recently used your BIDseq workflow and analysis pipeline - it is very straightforward to use, thank you for making it easily accessible!

I had one question about the filtering aspect of the pipeline. From what I can tell, the filter_sites contain sites that would be filtered out by the filter parameters published in Dai et al 2022 ((1) deletion rate above 5% (with deletion count above five in BID-seq libraries); (2) deletion rate below 1% in ‘Input’ libraries; (3) total reads coverage depth above 20 in both BID-seq and ‘Input’ libraries; (4) deletion rate above 1.5-fold over background in any given sequence motif (defined as the deletion rates detected from RNA probes containing 0% Ψ,). Would you recommend to use those parameters to further filter the output sites?

Thanks!

Low mapping ratio and lost reads after realigngap and filter

Hi y9c!
I am trying your pseudoU-BIDseq pipeline. I found your workflow is very efficient and your coding is perfect.Nice work!
I have a question here. when I ran the pipeline with the mRNA samples from Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution, I found the mapping ratio for genome is very low(~30% for unique mapping and ~6 % for multimapping), and I have lost lots of reads after realignGap and samtools calmd, samtools view -e '[NM]<=5 && [NM]/(qlen-sclen)<=0.1', the average of lost reads is about 40%.
I wonder is this correct and what can I do?
Thanks you!

samFilter: /lib64/libc.so.6: version `GLIBC_2.28' not found

Hi, may I ask what's the dependency for samFilter? The following error shows when executing the samFilter file under bin directory.
/lib64/libc.so.6: version GLIBC_2.28' not found (required by /users/ludwig/ebu571/ebu571/BIDseq/pseudoU-BIDseq-main/bin/samFilter) /lib64/libc.so.6: version GLIBC_2.18' not found (required by /users/ludwig/ebu571/ebu571/BIDseq/pseudoU-BIDseq-main/bin/samFilter)
/lib64/libc.so.6: version `GLIBC_2.25' not found (required by /users/ludwig/ebu571/ebu571/BIDseq/pseudoU-BIDseq-main/bin/samFilter).
Thanks.

automatic program termination

Hi y9c!

I encountered two instances of automatic program termination. My command is nohup apptainer run docker://y9ch/bidseq & in the terminal. And I always keep the terminal on. The attatchment is the two log files.
2024-01-11T111942.665433.snakemake.log
2024-01-12T003946.169422.snakemake.log

I wonder what can I do, thank you so much!

rcFastq

hello, i run you docker,
apptainer run -B /workplace bidseq_latest.sif

i produce error: /pipeline/bin/rcFastq: /lib/x86_64-linux-gnu/libc.so.6: version `GLIBC_2.34' not found (required by /pipeline/bin/rcFastq)

can you update this glibc?

--bind error

Hello y9c.

I'm trying to run the pipeline but it gives me an error. At the first run, the program suggested me to run again with the corresponding paths in the --bind arg, but even after doing so the error shows up again. I'm not sure if I'm missing something, or perhaps this a pipeline bug or even something related just to apptainer. I let you all the information I gathered:

Command:
apptainer -d run --bind /data/resources/refgenome/gencode_45/star_index,/data2/ane_chicago/small_RNA_pU,/data/resources/refgenome/gencode_45 bidseq_latest.sif

Input YAML file:

reference:
  genes:
    fa: ./myRNA.fa
  genome:
    fa: /data/resources/refgenome/gencode_45/GRCh38.p14.genome.fa
    star: /data/resources/refgenome/gencode_45/star_index/

cores: 8

samples:
  CHe-HS-24S-pU-AOG-7-0:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-7-0_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-7-0_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: false
  CHe-HS-24S-pU-AOG-7-1:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-7-1_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-7-1_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: true
  CHe-HS-24S-pU-AOG-8-0:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-8-0_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-8-0_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: false
  CHe-HS-24S-pU-AOG-8-1:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-8-1_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-8-1_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: true
  CHe-HS-24S-pU-AOG-9-0:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-9-0_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-9-0_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: false
  CHe-HS-24S-pU-AOG-9-1:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-9-1_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-9-1_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: true
  CHe-HS-24S-pU-AOG-10-0:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-10-0_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-10-0_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: false
  CHe-HS-24S-pU-AOG-10-1:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-10-1_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-10-1_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: true
  CHe-HS-24S-pU-AOG-11-0:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-11-0_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-11-0_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: false
  CHe-HS-24S-pU-AOG-11-1:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-11-1_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-11-1_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: true
  CHe-HS-24S-pU-AOG-12-0:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-12-0_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-12-0_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: false
  CHe-HS-24S-pU-AOG-12-1:
    data:
      - R1: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-12-1_S1_R1_001.fastq.gz
        R2: /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-12-1_S1_R2_001.fastq.gz
    group: ane_small_RNA_pU
    treated: true

stdout:

 ██╗ ██╗ ██╗      ██████╗ ██╗██████╗ ███████╗███████╗ ██████╗
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 ╚████████╔╝╚════╝██╔══██╗██║██║  ██║╚════██║██╔══╝  ██║▄▄ ██║
  ╚══██║══╝       ██████╔╝██║██████╔╝███████║███████╗╚██████╔╝
     ╚═╝          ╚═════╝ ╚═╝╚═════╝ ╚══════╝╚══════╝ ╚══▀▀═╝

    ██████╗ ██╗██████╗ ███████╗██╗     ██╗███╗   ██╗███████╗
    ██╔══██╗██║██╔══██╗██╔════╝██║     ██║████╗  ██║██╔════╝
    ██████╔╝██║██████╔╝█████╗  ██║     ██║██╔██╗ ██║█████╗
    ██╔═══╝ ██║██╔═══╝ ██╔══╝  ██║     ██║██║╚██╗██║██╔══╝
    ██║     ██║██║     ███████╗███████╗██║██║ ╚████║███████╗
    ╚═╝     ╚═╝╚═╝     ╚══════╝╚══════╝╚═╝╚═╝  ╚═══╝╚══════╝

Please add --bind /data/resources/refgenome/gencode_45/star_index,/data2/ane_chicago/small_RNA_pU,/data/resources/refgenome/gencode_45 right after 'apptainer run'
READ DEBUG LOG AT BIDSEQ_LOG_2024-06-06-094218.txt
❌ Jobs exit with error!`

BIDSEQ_LOG:

Building DAG of jobs...
MissingInputException in rule join_pairend_reads  in line 113 of /pipeline/Snakefile:
Missing input files for rule join_pairend_reads:
    output: .tmp/merged_reads/CHe-HS-24S-pU-AOG-7-1_run1.fq.gz, report_reads/joining/CHe-HS-24S-pU-AOG-7-1_run1.fastp.html, report_reads/joining/CHe-HS-24S-pU-AOG-7-1_run1.fastp.json
    wildcards: sample=CHe-HS-24S-pU-AOG-7-1, rn=run1
    affected files:
        /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-7-1_S1_R1_001.fastq.gz
        /data2/ane_chicago/small_RNA_pU/CHe-HS-24S-pU-AOG-7-1_S1_R2_001.fastq.gz

stderr:

DEBUG   [U=1000,P=2967986] persistentPreRun()            Apptainer version: 1.3.1-1.el8
DEBUG   [U=1000,P=2967986] persistentPreRun()            Parsing configuration file /etc/apptainer/apptainer.conf
DEBUG   [U=1000,P=2967986] SetBinaryPath()               Setting binary path to /usr/libexec/apptainer/bin:/home/sergi/.local/bin:/home/sergi/bin:/opt/miniconda3/condabin:/home/sergi/.sdkman/candidates/java/current/bin:/data/genotools/google-cloud-sdk/bin:/home/sergi/.cargo/bin:/home/sergi/.local/bin:/home/sergi/bin:/usr/share/Modules/bin:/usr/local/bin:/usr/bin:/usr/local/sbin:/usr/sbin:/usr/local/sbin:/usr/local/bin:/usr/sbin:/usr/bin:/sbin:/bin
DEBUG   [U=1000,P=2967986] SetBinaryPath()               Using that path for all binaries
DEBUG   [U=1000,P=2967986] handleConfDir()               /home/sergi/.apptainer already exists. Not creating.
DEBUG   [U=1000,P=2967986] setUmask()                    Saving umask 0002 for propagation into container
VERBOSE [U=1000,P=2967986] useSuid()                     Unprivileged installation: using user namespace
DEBUG   [U=1000,P=2967986] checkEncryptionKey()          Checking for encrypted system partition
DEBUG   [U=1000,P=2967986] Init()                        Image format detection
DEBUG   [U=1000,P=2967986] Init()                        Check for sandbox image format
DEBUG   [U=1000,P=2967986] Init()                        sandbox format initializer returned: not a directory image
DEBUG   [U=1000,P=2967986] Init()                        Check for sif image format
DEBUG   [U=1000,P=2967986] Init()                        sif image format detected
DEBUG   [U=1000,P=2967986] setCgroups()                  Recording rootless XDG_RUNTIME_DIR / DBUS_SESSION_BUS_ADDRESS
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding CONDA_SHLVL environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding LC_ALL environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding LS_COLORS environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding CONDA_EXE environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding SSH_CONNECTION environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding MODULES_RUN_QUARANTINE environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding LANG environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding HISTCONTROL environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding HOSTNAME environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding OLDPWD environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding SDKMAN_CANDIDATES_API environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding JAVA_HOME environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding _CE_M environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding which_declare environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding XDG_SESSION_ID environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding MODULES_CMD environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding USER environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding PWD environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding SSH_ASKPASS environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding CONDA_PYTHON_EXE environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding LC_CTYPE environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding SSH_CLIENT environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding TMUX environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding XDG_DATA_DIRS environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding _CE_CONDA environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding LOADEDMODULES environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding SDKMAN_DIR environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding SSH_TTY environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding MAIL environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding TERM environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding SHELL environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding SDKMAN_CANDIDATES_DIR environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding TMUX_PANE environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding SHLVL environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding MANPATH environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding MODULEPATH environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding LOGNAME environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding DBUS_SESSION_BUS_ADDRESS environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding XDG_RUNTIME_DIR environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding MODULEPATH_modshare environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding DEBUGINFOD_URLS environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding MODULESHOME environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding HISTSIZE environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding SDKMAN_PLATFORM environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding LESSOPEN environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding BASH_FUNC_which%% environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding BASH_FUNC_module%% environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding BASH_FUNC__module_raw%% environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding BASH_FUNC_switchml%% environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding BASH_FUNC_scl%% environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding BASH_FUNC_ml%% environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding _ environment variable
VERBOSE [U=1000,P=2967986] SetContainerEnv()             Not forwarding APPTAINER_DEBUG environment variable
DEBUG   [U=1000,P=2967986] SetContainerEnv()             Forwarding USER_PATH environment variable
VERBOSE [U=1000,P=2967986] SetContainerEnv()             Setting HOME=/home/sergi
VERBOSE [U=1000,P=2967986] SetContainerEnv()             Setting PATH=/usr/local/sbin:/usr/local/bin:/usr/sbin:/usr/bin:/sbin:/bin
DEBUG   [U=1000,P=2967986] findOnPath()                  Found "squashfuse_ll" at "/usr/libexec/apptainer/bin/squashfuse_ll"
DEBUG   [U=1000,P=2967986] findOnPath()                  Found "fuse2fs" at "/usr/libexec/apptainer/bin/fuse2fs"
DEBUG   [U=1000,P=2967986] findOnPath()                  Found "fuse-overlayfs" at "/usr/libexec/apptainer/bin/fuse-overlayfs"
DEBUG   [U=1000,P=2967986] findOnPath()                  Found "gocryptfs" at "/usr/libexec/apptainer/bin/gocryptfs"
DEBUG   [U=1000,P=2967986] InitImageDrivers()            squashfuse_ll supports -o uid
DEBUG   [U=1000,P=2967986] InitImageDrivers()            Setting ImageDriver to fuseapps
DEBUG   [U=1000,P=2967986] init()                        Use starter binary /usr/libexec/apptainer/bin/starter
VERBOSE [U=1000,P=2967986]  print()                       Set messagelevel to: 5
VERBOSE [U=1000,P=2967986]  init()                        Starter initialization
VERBOSE [U=1000,P=2967986]  is_suid()                     Check if we are running as setuid: 0
DEBUG   [U=1000,P=2967986]  read_engine_config()          Read engine configuration
DEBUG   [U=1000,P=2967986]  init()                        Wait completion of stage1
DEBUG   [U=1000,P=2968000]  set_parent_death_signal()     Set parent death signal to 9
VERBOSE [U=1000,P=2968000]  init()                        Spawn stage 1
DEBUG   [U=1000,P=2968000] func1()                       executablePath is /usr/libexec/apptainer/bin/starter
DEBUG   [U=1000,P=2968000] func1()                       starter was not relocated from /usr/libexec
DEBUG   [U=1000,P=2968000] func1()                       Install prefix is /usr
DEBUG   [U=1000,P=2968000] startup()                     apptainer runtime engine selected
VERBOSE [U=1000,P=2968000] startup()                     Execute stage 1
DEBUG   [U=1000,P=2968000] StageOne()                    Entering stage 1
DEBUG   [U=1000,P=2968000] SetuidMountAllowed()          Kernel squashfs mount not allowed because ECL not activated
DEBUG   [U=1000,P=2968000] findOnPath()                  Found "squashfuse_ll" at "/usr/libexec/apptainer/bin/squashfuse_ll"
DEBUG   [U=1000,P=2968000] findOnPath()                  Found "fuse2fs" at "/usr/libexec/apptainer/bin/fuse2fs"
DEBUG   [U=1000,P=2968000] findOnPath()                  Found "fuse-overlayfs" at "/usr/libexec/apptainer/bin/fuse-overlayfs"
DEBUG   [U=1000,P=2968000] findOnPath()                  Found "gocryptfs" at "/usr/libexec/apptainer/bin/gocryptfs"
DEBUG   [U=1000,P=2968000] InitImageDrivers()            squashfuse_ll supports -o uid
DEBUG   [U=1000,P=2968000] InitImageDrivers()            Setting ImageDriver to fuseapps
DEBUG   [U=1000,P=2968000] prepareAutofs()               Found "/proc/sys/fs/binfmt_misc" as autofs mount point
DEBUG   [U=1000,P=2968000] prepareAutofs()               Could not keep file descriptor for user bind path /data/resources/refgenome/gencode_45/star_index: no mount point
DEBUG   [U=1000,P=2968000] prepareAutofs()               Could not keep file descriptor for user bind path /data2/ane_chicago/small_RNA_pU: no mount point
DEBUG   [U=1000,P=2968000] prepareAutofs()               Could not keep file descriptor for user bind path /data/resources/refgenome/gencode_45: no mount point
DEBUG   [U=1000,P=2968000] prepareAutofs()               Could not keep file descriptor for bind path /etc/localtime: no mount point
DEBUG   [U=1000,P=2968000] prepareAutofs()               Could not keep file descriptor for bind path /etc/hosts: no mount point
DEBUG   [U=1000,P=2968000] prepareAutofs()               Could not keep file descriptor for home directory /home/sergi: no mount point
DEBUG   [U=1000,P=2968000] prepareAutofs()               Could not keep file descriptor for current working directory /data/cajon/tommaso_22_05_2024_B: no mount point
DEBUG   [U=1000,P=2968000] Init()                        Image format detection
DEBUG   [U=1000,P=2968000] Init()                        Check for sandbox image format
DEBUG   [U=1000,P=2968000] Init()                        sandbox format initializer returned: not a directory image
DEBUG   [U=1000,P=2968000] Init()                        Check for sif image format
DEBUG   [U=1000,P=2968000] Init()                        sif image format detected
DEBUG   [U=1000,P=2968000] setSessionLayer()             Using overlay because it is not disabled
DEBUG   [U=1000,P=2968000] PrepareConfig()               image driver is fuseapps
VERBOSE [U=1000,P=2967986]  wait_child()                  stage 1 exited with status 0
DEBUG   [U=1000,P=2967986]  cleanup_fd()                  Close file descriptor 4
DEBUG   [U=1000,P=2967986]  cleanup_fd()                  Close file descriptor 5
DEBUG   [U=1000,P=2967986]  cleanup_fd()                  Close file descriptor 6
DEBUG   [U=1000,P=2967986]  init()                        Set child signal mask
DEBUG   [U=1000,P=2967986]  init()                        Create socketpair for master communication channel
DEBUG   [U=1000,P=2967986]  init()                        Create RPC socketpair for communication between stage 2 and RPC server
VERBOSE [U=1000,P=2967986]  user_namespace_init()         Create user namespace
VERBOSE [U=1000,P=2967986]  create_namespace()            Create user namespace
VERBOSE [U=65534,P=2967986]  init()                        Spawn master process
DEBUG   [U=65534,P=2967986]  setup_userns_mappings()       Write deny to setgroups file
DEBUG   [U=65534,P=2967986]  setup_userns_mappings()       Write to GID map
DEBUG   [U=65534,P=2967986]  setup_userns_mappings()       Write to UID map
DEBUG   [U=1000,P=2968007]  set_parent_death_signal()     Set parent death signal to 9
VERBOSE [U=1000,P=2968007]  create_namespace()            Create mount namespace
VERBOSE [U=1000,P=2967986]  enter_namespace()             Entering in mount namespace
DEBUG   [U=1000,P=2967986]  enter_namespace()             Opening namespace file ns/mnt
VERBOSE [U=1000,P=2968007]  create_namespace()            Create mount namespace
VERBOSE [U=1000,P=2968008]  init()                        Spawn RPC server
DEBUG   [U=1000,P=2967986] func1()                       executablePath is /usr/libexec/apptainer/bin/starter
DEBUG   [U=1000,P=2967986] func1()                       starter was not relocated from /usr/libexec
DEBUG   [U=1000,P=2967986] func1()                       Install prefix is /usr
DEBUG   [U=1000,P=2968008] func1()                       executablePath is /usr/libexec/apptainer/bin/starter
DEBUG   [U=1000,P=2968008] func1()                       starter was not relocated from /usr/libexec
DEBUG   [U=1000,P=2968008] func1()                       Install prefix is /usr
DEBUG   [U=1000,P=2967986] startup()                     apptainer runtime engine selected
VERBOSE [U=1000,P=2967986] startup()                     Execute master process
DEBUG   [U=1000,P=2968008] startup()                     apptainer runtime engine selected
VERBOSE [U=1000,P=2968008] startup()                     Serve RPC requests
DEBUG   [U=1000,P=2967986] findOnPath()                  Found "squashfuse_ll" at "/usr/libexec/apptainer/bin/squashfuse_ll"
DEBUG   [U=1000,P=2967986] findOnPath()                  Found "fuse2fs" at "/usr/libexec/apptainer/bin/fuse2fs"
DEBUG   [U=1000,P=2967986] findOnPath()                  Found "fuse-overlayfs" at "/usr/libexec/apptainer/bin/fuse-overlayfs"
DEBUG   [U=1000,P=2967986] findOnPath()                  Found "gocryptfs" at "/usr/libexec/apptainer/bin/gocryptfs"
DEBUG   [U=1000,P=2967986] InitImageDrivers()            squashfuse_ll supports -o uid
DEBUG   [U=1000,P=2967986] InitImageDrivers()            Setting ImageDriver to fuseapps
DEBUG   [U=1000,P=2967986] setupSessionLayout()          Using Layer system: overlay
DEBUG   [U=1000,P=2967986] setupOverlayLayout()          Creating overlay SESSIONDIR layout
DEBUG   [U=1000,P=2967986] addRootfsMount()              Mount rootfs in read-only mode
DEBUG   [U=1000,P=2967986] addRootfsMount()              Image type is 4096
DEBUG   [U=1000,P=2967986] addRootfsMount()              Mounting block [squashfs] image: /data/cajon/tommaso_22_05_2024_B/bidseq_latest.sif
DEBUG   [U=1000,P=2967986] addKernelMount()              Checking configuration file for 'mount proc'
DEBUG   [U=1000,P=2967986] addKernelMount()              Adding proc to mount list
VERBOSE [U=1000,P=2967986] addKernelMount()              Default mount: /proc:/proc
DEBUG   [U=1000,P=2967986] addKernelMount()              Checking configuration file for 'mount sys'
DEBUG   [U=1000,P=2967986] addKernelMount()              Adding sysfs to mount list
VERBOSE [U=1000,P=2967986] addKernelMount()              Default mount: /sys:/sys
DEBUG   [U=1000,P=2967986] addDevMount()                 Checking configuration file for 'mount dev'
DEBUG   [U=1000,P=2967986] addDevMount()                 Adding dev to mount list
VERBOSE [U=1000,P=2967986] addDevMount()                 Default mount: /dev:/dev
DEBUG   [U=1000,P=2967986] addHostMount()                Not mounting host file systems per configuration
VERBOSE [U=1000,P=2967986] addBindsMount()               Found 'bind path' = /etc/localtime, /etc/localtime
VERBOSE [U=1000,P=2967986] addBindsMount()               Found 'bind path' = /etc/hosts, /etc/hosts
DEBUG   [U=1000,P=2967986] addHomeStagingDir()           Staging home directory (/home/sergi) at /var/lib/apptainer/mnt/session/home/sergi
DEBUG   [U=1000,P=2967986] addHomeMount()                Adding home directory mount [/var/lib/apptainer/mnt/session/home/sergi:/home/sergi] to list using layer: overlay
DEBUG   [U=1000,P=2967986] addUserbindsMount()           Adding /data/resources/refgenome/gencode_45/star_index to mount list
DEBUG   [U=1000,P=2967986] addUserbindsMount()           Adding /data2/ane_chicago/small_RNA_pU to mount list
DEBUG   [U=1000,P=2967986] addUserbindsMount()           Adding /data/resources/refgenome/gencode_45 to mount list
DEBUG   [U=1000,P=2967986] addTmpMount()                 Checking for 'mount tmp' in configuration file
DEBUG   [U=1000,P=2967986] addScratchMount()             Not mounting scratch directory: Not requested
DEBUG   [U=1000,P=2967986] addLibsMount()                Checking for 'user bind control' in configuration file
DEBUG   [U=1000,P=2967986] addFilesMount()               Checking for 'user bind control' in configuration file
DEBUG   [U=1000,P=2967986] addResolvConfMount()          Adding /etc/resolv.conf to mount list
VERBOSE [U=1000,P=2967986] addResolvConfMount()          Default mount: /etc/resolv.conf:/etc/resolv.conf
DEBUG   [U=1000,P=2967986] addHostnameMount()            Skipping hostname mount, not virtualizing UTS namespace on user request
DEBUG   [U=1000,P=2967986] create()                      Mount all
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting tmpfs to /var/lib/apptainer/mnt/session
DEBUG   [U=1000,P=2967986] func2()                       Starting image driver fuseapps
DEBUG   [U=1000,P=2967986] Mount()                       Executing /usr/libexec/apptainer/bin/squashfuse_ll -f -o allow_other,ro,uid=1000,gid=1000,offset=36864 /proc/self/fd/3 /var/lib/apptainer/mnt/session/rootfs
DEBUG   [U=1000,P=2967986] Mount()                       /var/lib/apptainer/mnt/session/rootfs mounted in 25ms
DEBUG   [U=1000,P=2967986] createCwdDir()                Using /data/cajon/tommaso_22_05_2024_B as current working directory
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting overlay to /var/lib/apptainer/mnt/session/final
DEBUG   [U=1000,P=2967986] mountGeneric()                Unmounting and remounting overlay
DEBUG   [U=1000,P=2967986] mountGeneric()                Remounting /var/lib/apptainer/mnt/session/final
DEBUG   [U=1000,P=2967986] setPropagationMount()         Set RPC mount propagation flag to SLAVE
VERBOSE [U=1000,P=2967986] Passwd()                      Checking for template passwd file: /var/lib/apptainer/mnt/session/rootfs/etc/passwd
VERBOSE [U=1000,P=2967986] Passwd()                      Creating passwd content
VERBOSE [U=1000,P=2967986] Passwd()                      Creating template passwd file and injecting user data: /var/lib/apptainer/mnt/session/rootfs/etc/passwd
DEBUG   [U=1000,P=2967986] addIdentityMount()            Adding /etc/passwd to mount list
VERBOSE [U=1000,P=2967986] addIdentityMount()            Default mount: /etc/passwd:/etc/passwd
VERBOSE [U=1000,P=2967986] Group()                       Checking for template group file: /var/lib/apptainer/mnt/session/rootfs/etc/group
VERBOSE [U=1000,P=2967986] Group()                       Creating group content
DEBUG   [U=1000,P=2967986] addIdentityMount()            Adding /etc/group to mount list
VERBOSE [U=1000,P=2967986] addIdentityMount()            Default mount: /etc/group:/etc/group
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /dev to /var/lib/apptainer/mnt/session/final/dev
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /etc/localtime to /var/lib/apptainer/mnt/session/final/etc/localtime
DEBUG   [U=1000,P=2967986] mountGeneric()                Remounting /var/lib/apptainer/mnt/session/final/etc/localtime
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /etc/hosts to /var/lib/apptainer/mnt/session/final/etc/hosts
DEBUG   [U=1000,P=2967986] mountGeneric()                Remounting /var/lib/apptainer/mnt/session/final/etc/hosts
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /proc to /var/lib/apptainer/mnt/session/final/proc
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /sys to /var/lib/apptainer/mnt/session/final/sys
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /home/sergi to /var/lib/apptainer/mnt/session/home/sergi
DEBUG   [U=1000,P=2967986] mountGeneric()                Remounting /var/lib/apptainer/mnt/session/home/sergi
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /var/lib/apptainer/mnt/session/home/sergi to /var/lib/apptainer/mnt/session/final/home/sergi
DEBUG   [U=1000,P=2967986] func1()                       Container /tmp resolves to "/tmp"
DEBUG   [U=1000,P=2967986] func1()                       Container /var/tmp resolves to "/var/tmp"
VERBOSE [U=1000,P=2967986] func1()                       Default mount: /tmp:/tmp
VERBOSE [U=1000,P=2967986] func1()                       Default mount: /var/tmp:/var/tmp
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /tmp to /var/lib/apptainer/mnt/session/final/tmp
DEBUG   [U=1000,P=2967986] mountGeneric()                Remounting /var/lib/apptainer/mnt/session/final/tmp
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /var/tmp to /var/lib/apptainer/mnt/session/final/var/tmp
DEBUG   [U=1000,P=2967986] mountGeneric()                Remounting /var/lib/apptainer/mnt/session/final/var/tmp
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /var/lib/apptainer/mnt/session/etc/resolv.conf to /var/lib/apptainer/mnt/session/final/etc/resolv.conf
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /var/lib/apptainer/mnt/session/etc/passwd to /var/lib/apptainer/mnt/session/final/etc/passwd
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /var/lib/apptainer/mnt/session/etc/group to /var/lib/apptainer/mnt/session/final/etc/group
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /data2/ane_chicago/small_RNA_pU to /var/lib/apptainer/mnt/session/final/data2/ane_chicago/small_RNA_pU
DEBUG   [U=1000,P=2967986] mountGeneric()                Remounting /var/lib/apptainer/mnt/session/final/data2/ane_chicago/small_RNA_pU
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /data/resources/refgenome/gencode_45 to /var/lib/apptainer/mnt/session/final/data/resources/refgenome/gencode_45
DEBUG   [U=1000,P=2967986] mountGeneric()                Remounting /var/lib/apptainer/mnt/session/final/data/resources/refgenome/gencode_45
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /data/resources/refgenome/gencode_45/star_index to /var/lib/apptainer/mnt/session/final/data/resources/refgenome/gencode_45/star_index
DEBUG   [U=1000,P=2967986] mountGeneric()                Remounting /var/lib/apptainer/mnt/session/final/data/resources/refgenome/gencode_45/star_index
DEBUG   [U=1000,P=2967986] isMounted()                   Checking if /var/lib/apptainer/mnt/session/final/data/cajon/tommaso_22_05_2024_B is already mounted
DEBUG   [U=1000,P=2967986] mountGeneric()                Mounting /data/cajon/tommaso_22_05_2024_B to /var/lib/apptainer/mnt/session/final/data/cajon/tommaso_22_05_2024_B
DEBUG   [U=1000,P=2967986] mountGeneric()                Remounting /var/lib/apptainer/mnt/session/final/data/cajon/tommaso_22_05_2024_B
DEBUG   [U=1000,P=2967986] create()                      Chroot into /var/lib/apptainer/mnt/session/final
DEBUG   [U=1000,P=2968008] Chroot()                      Hold reference to host / directory
DEBUG   [U=1000,P=2968008] Chroot()                      Called pivot_root on /var/lib/apptainer/mnt/session/final
DEBUG   [U=1000,P=2968008] Chroot()                      Change current directory to host / directory
DEBUG   [U=1000,P=2968008] Chroot()                      Apply slave mount propagation for host / directory
DEBUG   [U=1000,P=2968008] Chroot()                      Called unmount(/, syscall.MNT_DETACH)
DEBUG   [U=1000,P=2968008] Chroot()                      Changing directory to / to avoid getpwd issues
DEBUG   [U=1000,P=2967986] create()                      Chdir into / to avoid errors
VERBOSE [U=1000,P=2968007]  wait_child()                  rpc server exited with status 0
DEBUG   [U=1000,P=2968007]  init()                        Set container privileges
DEBUG   [U=1000,P=2968007]  apply_privileges()            Effective capabilities:   0x0000000000000000
DEBUG   [U=1000,P=2968007]  apply_privileges()            Permitted capabilities:   0x0000000000000000
DEBUG   [U=1000,P=2968007]  apply_privileges()            Bounding capabilities:    0x0000000000000000
DEBUG   [U=1000,P=2968007]  apply_privileges()            Inheritable capabilities: 0x0000000000000000
DEBUG   [U=1000,P=2968007]  apply_privileges()            Ambient capabilities:     0x0000000000000000
DEBUG   [U=1000,P=2968007]  apply_privileges()            Set user ID to 1000
DEBUG   [U=1000,P=2968007]  set_parent_death_signal()     Set parent death signal to 9
DEBUG   [U=1000,P=2968007] func1()                       executablePath is /usr/libexec/apptainer/bin/starter
DEBUG   [U=1000,P=2968007] func1()                       executablePath does not exist, assuming default prefix
DEBUG   [U=1000,P=2968007] startup()                     apptainer runtime engine selected
VERBOSE [U=1000,P=2968007] startup()                     Execute stage 2
DEBUG   [U=1000,P=2968007] StageTwo()                    Entering stage 2
DEBUG   [U=1000,P=2968007] StartProcess()                Setting umask in container to 0002
DEBUG   [U=1000,P=2968007] func1()                       Not exporting "BASH_FUNC__module_raw%%" to container environment: invalid key
DEBUG   [U=1000,P=2968007] func1()                       Not exporting "BASH_FUNC_ml%%" to container environment: invalid key
DEBUG   [U=1000,P=2968007] func1()                       Not exporting "BASH_FUNC_module%%" to container environment: invalid key
DEBUG   [U=1000,P=2968007] func1()                       Not exporting "BASH_FUNC_scl%%" to container environment: invalid key
DEBUG   [U=1000,P=2968007] func1()                       Not exporting "BASH_FUNC_switchml%%" to container environment: invalid key
DEBUG   [U=1000,P=2968007] func1()                       Not exporting "BASH_FUNC_which%%" to container environment: invalid key
DEBUG   [U=1000,P=2968007] sylogBuiltin()                Sourcing /.singularity.d/env/01-base.sh
DEBUG   [U=1000,P=2968007] sylogBuiltin()                Sourcing /.singularity.d/env/10-docker2singularity.sh
DEBUG   [U=1000,P=2968007] sylogBuiltin()                Sourcing /.singularity.d/env/90-environment.sh
DEBUG   [U=1000,P=2968007] sylogBuiltin()                Sourcing /.singularity.d/env/94-appsbase.sh
DEBUG   [U=1000,P=2968007] sylogBuiltin()                Sourcing /.singularity.d/env/95-apps.sh
DEBUG   [U=1000,P=2968007] sylogBuiltin()                Sourcing /.singularity.d/env/99-base.sh
DEBUG   [U=1000,P=2968007] sylogBuiltin()                Sourcing /.singularity.d/env/99-runtimevars.sh
DEBUG   [U=1000,P=2968007] sylogBuiltin()                Running action command run
DEBUG   [U=1000,P=2967986] PostStartProcess()            Post start process
bash: warning: setlocale: LC_ALL: cannot change locale (en_US.UTF-8)
DEBUG   [U=1000,P=2967986] CleanupContainer()            Cleanup container
DEBUG   [U=1000,P=2967986] umount()                      Umount /var/lib/apptainer/mnt/session/final
DEBUG   [U=1000,P=2967986] umount()                      Umount /var/lib/apptainer/mnt/session/rootfs
DEBUG   [U=1000,P=2967986] stop()                        Waiting for squashfuse_ll pid 2968020 to exit
DEBUG   [U=1000,P=2967986] Master()                      Child exited with exit status 0

I tried also with root permissions, which I have access to.

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