Process MRS:CALL_MRS (24R3:genome_matrix.tsv) terminated with an error exit status (127)

[MagdalenaWinklhofer]

Hi,

I am working with WGBS data from fish. I aligned my short reads to the genome with bismark and would like to use Methylscore to investigate the data further. But when I start the script, it runs for 82h and stops with an error.

Here is the error inside the slurm output: WARN: Killing running tasks (2)

executor > local (6651) [9a/2fba66] process > CONSENSUS:SORT_BAM (N7) [100%] 12 of 12 ✔ [- ] process > CONSENSUS:MERGE_BAM - [ec/6d8e6c] process > CONSENSUS:DEDUPLICATE (N2) [100%] 13 of 13, failed:... [10/8cdd18] process > CONSENSUS:READ_STATISTICS (N3) [100%] 12 of 12 ✔ [8e/abd7ec] process > CONSENSUS:SPLIT_BAM (N2:sca... [100%] 3144 of 3144 ✔ [9a/7f3c97] process > CONSENSUS:METHYLDACKEL (N2:... [100%] 3144 of 3144 ✔ [4b/d7b8b5] process > CONSENSUS:MATRIX:BUILD (sca... [100%] 262 of 262 ✔ [81/c3f901] process > SAMPLESHEET:GENERATE (genom... [100%] 1 of 1 ✔ [79/84e155] process > MRS:CALL_MRS (24R3:genome_m... [ 85%] 61 of 71, failed:... [- ] process > MRS:MR_STATISTICS - [- ] process > MRS:SPLIT_MRS - [- ] process > DMRS:INDEX (genome_matrix.tsv) - [- ] process > DMRS:CALL_DMRS - [- ] process > DMRS:MERGE_DMRS - [33/04488b] NOTE: Process MRS:CALL_MRS (24R3:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [ac/f0a95a] NOTE: Process MRS:CALL_MRS (24R8:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [ea/4f40be] NOTE: Process MRS:CALL_MRS (24R7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [11/e2b67d] NOTE: Process MRS:CALL_MRS (A4:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [20/87fb25] NOTE: Process MRS:CALL_MRS (A7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [85/972eba] NOTE: Process MRS:CALL_MRS (24R2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [ad/391f59] NOTE: Process MRS:CALL_MRS (A1:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [fb/e5e9b9] NOTE: Process MRS:CALL_MRS (A2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [d8/8418d5] NOTE: Process MRS:CALL_MRS (N2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [82/7e32af] NOTE: Process MRS:CALL_MRS (N1:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [56/c60238] NOTE: Process MRS:CALL_MRS (N3:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [b9/9f1b5e] NOTE: Process MRS:CALL_MRS (N7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (1) [0b/ef14ba] NOTE: Process MRS:CALL_MRS (24R8:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [2e/004801] NOTE: Process MRS:CALL_MRS (24R3:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [7b/69ad38] NOTE: Process MRS:CALL_MRS (24R7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [01/f4409e] NOTE: Process MRS:CALL_MRS (A4:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [a7/924094] NOTE: Process MRS:CALL_MRS (A7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [92/183f2a] NOTE: Process MRS:CALL_MRS (24R2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [4e/b7b1bb] NOTE: Process MRS:CALL_MRS (A1:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [b8/e41eab] NOTE: Process MRS:CALL_MRS (A2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [3e/c9c39d] NOTE: Process MRS:CALL_MRS (N2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [8f/2a64f7] NOTE: Process MRS:CALL_MRS (N1:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [c5/7f9bd9] NOTE: Process MRS:CALL_MRS (N3:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [d0/db72dd] NOTE: Process MRS:CALL_MRS (N7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (2) [f6/c5d1ed] NOTE: Process MRS:CALL_MRS (24R8:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [ff/3cc3d2] NOTE: Process MRS:CALL_MRS (24R3:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [43/d3d8c9] NOTE: Process MRS:CALL_MRS (24R7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [b8/173525] NOTE: Process MRS:CALL_MRS (A4:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [51/f9ddad] NOTE: Process MRS:CALL_MRS (A7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [ee/9c5aff] NOTE: Process MRS:CALL_MRS (24R2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [8e/74e866] NOTE: Process MRS:CALL_MRS (A1:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [34/59ad95] NOTE: Process MRS:CALL_MRS (A2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [12/ccd90f] NOTE: Process MRS:CALL_MRS (N1:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [0f/e03d3a] NOTE: Process MRS:CALL_MRS (N2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [f3/486198] NOTE: Process MRS:CALL_MRS (N3:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [8c/9dc36e] NOTE: Process MRS:CALL_MRS (24R8:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [3e/0e8e31] NOTE: Process MRS:CALL_MRS (N7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (3) [66/9d35aa] NOTE: Process MRS:CALL_MRS (24R3:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [57/fd016b] NOTE: Process MRS:CALL_MRS (24R7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [64/731e96] NOTE: Process MRS:CALL_MRS (A7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [67/c98447] NOTE: Process MRS:CALL_MRS (A4:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [cf/9c2b28] NOTE: Process MRS:CALL_MRS (24R2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [62/ea1967] NOTE: Process MRS:CALL_MRS (A1:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [8e/a5356b] NOTE: Process MRS:CALL_MRS (A2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [81/3a9e9a] NOTE: Process MRS:CALL_MRS (N1:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [c4/244ad2] NOTE: Process MRS:CALL_MRS (N2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [d6/8a642e] NOTE: Process MRS:CALL_MRS (N3:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [8d/d928f8] NOTE: Process MRS:CALL_MRS (24R8:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) [81/660b7c] NOTE: Process MRS:CALL_MRS (N7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (4) [4a/61a6c4] NOTE: Process MRS:CALL_MRS (24R3:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) [7f/1a49bb] NOTE: Process MRS:CALL_MRS (24R7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) [f6/65494e] NOTE: Process MRS:CALL_MRS (A7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) [30/b5c9d4] NOTE: Process MRS:CALL_MRS (A4:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) [52/29ad8b] NOTE: Process MRS:CALL_MRS (24R2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) [fc/d8b98c] NOTE: Process MRS:CALL_MRS (A2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) [89/539882] NOTE: Process MRS:CALL_MRS (A1:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) [09/0b982d] NOTE: Process MRS:CALL_MRS (N1:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) [19/056bd9] NOTE: Process MRS:CALL_MRS (N2:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) [f0/ef8a25] NOTE: Process MRS:CALL_MRS (N7:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) [76/ea32d5] NOTE: Process MRS:CALL_MRS (N3:genome_matrix.tsv) terminated with an error exit status (127) -- Execution is retried (5) Error executing process > 'MRS:CALL_MRS (24R8:genome_matrix.tsv)'

Caused by: Process MRS:CALL_MRS (24R8:genome_matrix.tsv) terminated with an error exit status (127)

Command executed:

hmm_mrs \ -x 3 \ -y 24R8 \ -c 1 \ -o 24R8.MRs.bed \ -d 100 \ -i 30 \ -m 30 \ -t 0.1 \ \ -n 20 \ genome_matrix.tsv \ -p 24R8.hmm_params

Command exit status: 127

Command output: (empty)

Command error: INFO: Environment variable SINGULARITYENV_TMPDIR is set, but APPTAINERENV_TMPDIR is preferred INFO: Environment variable SINGULARITYENV_NXF_DEBUG is set, but APPTAINERENV_NXF_DEBUG is preferred .command.sh: line 3: hmm_mrs: command not found ps: error while loading shared libraries: libdl.so.2: cannot open shared object file: No such file or directory

Work dir: /cluster/work/users/magdalena/DMR/work/02/957bee5c3e81eed0696076585f586d

Tip: you can try to figure out what's wrong by changing to the process work dir and showing the script file named .command.sh

WARN: Failed to render execution report -- see the log file for details WARN: Failed to render execution timeline -- see the log file for details

As far as I understand, the program can’t find the genome_matrix.tsv file. But this file is created inside the pipeline (not by me). I can see in the work directory it created the directories “01mapping”(contains files), “02consensus”(contains files), and “04MRs”(is empty), but it did not create the directory “03matrix” which should contain the files “genome_matrix.tsv.gz” and “genome_matrix.tsv.gz.tbi”. I also understood that exit status 127 means that the current command could not be found.

Regarding the environmental variables, I only stated those in the .sh script itself: “ export SINGULARITY_BIND="/cluster/.../14_DMR" export APPTAINER_TMPDIR=/cluster/work/users/magdalena/DMR/ export NXF_HOME=/cluster/work/users/magdalena/DMR/.nextflow “

And those in the configuration file: “ process.container = '/cluster/work/users/magdalena/DMR/methylscore.sif' singularity.enabled = true singularity.cacheDir = '/cluster/work/users/magdalena/DMR/' trace.overwrite = true singularity.autoMounts = true dag.overwrite = true “

Should I rename the “APPTAINER_TMPDIR” to “APPTAINERENV_TMPDIR” and “NXF_HOME” to “APPTAINERENV_NXF_DEBUG”? Do you think that would solve the issue? Is that related to the “genome_matrix.tsv file” issue?

If you need more information, please let me know. Thank you very much for your help!