Errors with nextPolish.sh.e

[Gerlex89]

Hi all. I'm trying to polish an assembly from Flye for nanopore reads. However, I cannot make the command work as I receive errors that I cannot determine the source, or how to proceed. These errors appear also with the test data (nextPolish test_data/run.cfg).

Operating system Linux Mint 20.2 Uma (Ubuntu Focal base)

GCC gcc version 9.3.0 (Ubuntu 9.3.0-17ubuntu1~20.04)

Python Python 3.9.7

NextPolish nextPolish v1.4.0

Input files (https://nextpolish.readthedocs.io/en/latest/TUTORIAL.html#polishing-using-long-reads-only)

lgs.fofn: I have only one FASTQ file because I'm testing the tool to adapt it to CWL, proceeding with ls /path/to/fastq_runid.fastq > lgs.fofn

run.cfg: modified parts are commented

[General]
job_type = local
job_prefix = nextPolish
task = best
rewrite = yes
rerun = 3
parallel_jobs = 6
multithread_jobs = 5
genome = ./assembly.fasta # modified
genome_size = auto
workdir = ./01_rundir # modified, tried with different
polish_options = -p {multithread_jobs}

[lgs_option]
lgs_fofn = ./lgs.fofn
lgs_options = -min_read_len 1k -max_depth 100
lgs_minimap2_options = -x map-ont

Log

[183854 INFO] 2021-12-03 10:52:24 NextPolish start...
[183854 INFO] 2021-12-03 10:52:24 version:v1.4.0 logfile:pid183854.log.info
[183854 WARNING] 2021-12-03 10:52:24 Delete task: 5 due to missing lgs_fofn.
[183854 WARNING] 2021-12-03 10:52:24 Delete task: 5 due to missing lgs_fofn.
[183854 WARNING] 2021-12-03 10:52:24 Delete task: 6 due to missing hifi_fofn.
[183854 WARNING] 2021-12-03 10:52:24 Delete task: 6 due to missing hifi_fofn.
[183854 INFO] 2021-12-03 10:52:24 scheduled tasks:
[1, 2, 1, 2]
[183854 INFO] 2021-12-03 10:52:24 options: 
[183854 INFO] 2021-12-03 10:52:24 
rerun:                        3
rewrite:                      0
kill:                         None
cleantmp:                     0
use_drmaa:                    0
submit:                       None
job_type:                     local
sgs_unpaired:                 0
sgs_rm_nread:                 1
lgs_read_type:                
parallel_jobs:                6
align_threads:                5
check_alive:                  None
task:                         [1, 2, 1, 2]
job_id_regex:                 None
genome_size:                  18910
sgs_max_depth:                100
lgs_max_depth:                100
multithread_jobs:             5
lgs_max_read_len:             0
hifi_max_depth:               100
lgs_block_size:               500M
lgs_min_read_len:             1k
hifi_max_read_len:            0
polish_options:               -p 5
hifi_block_size:              500M
hifi_min_read_len:            1k
job_prefix:                   nextPolish
sgs_use_duplicate_reads:      0
lgs_minimap2_options:         -x map-ont
hifi_minimap2_options:        -x map-pb
sgs_block_size:               315166.6666666667
sgs_align_options:            bwa mem -p  -t 5
workdir:                      path/to/NextPolish.backup0
genome:                       path/to/flye-output/assembly.fasta
sgs_fofn:                     path/to/NextPolish.backup0/test.fofn
snp_phase:                    path/to/NextPolish.backup0/%02d.snp_phase
snp_valid:                    path/to/NextPolish.backup0/%02d.snp_valid
lgs_polish:                   path/to/NextPolish.backup0/%02d.lgs_polish
kmer_count:                   path/to/NextPolish.backup0/%02d.kmer_count
hifi_polish:                  path/to/NextPolish.backup0/%02d.hifi_polish
score_chain:                  path/to/NextPolish.backup0/%02d.score_chain
[183854 WARNING] 2021-12-03 10:52:24 mv path/to/NextPolish.backup0 to path/to/NextPolish.backup0.backup0
[183854 INFO] 2021-12-03 10:52:24 step 0 and task 1 start:
[183854 INFO] 2021-12-03 10:52:29 Total jobs: 3
[183854 INFO] 2021-12-03 10:52:29 Submitted jobID:[183883] jobCmd:[path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split1/nextPolish.sh] in the local_cycle.
[183883 CRITICAL] 2021-12-03 10:52:29 Command '/bin/sh path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split1/nextPolish.sh > path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split1/nextPolish.sh.e' returned non-zero exit status 127, error info: .
Traceback (most recent call last):
  File "path/to/NextPolish.backup0/./nextPolish", line 515, in <module>
  File "path/to/NextPolish.backup0/./nextPolish", line 369, in main
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 347, in start
    self._start()
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 371, in _start
    self.submit(job)
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 255, in submit
    _, stdout, _ = self.run(job.cmd)
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 291, in run
    log.critical("Command '%s' returned non-zero exit status %d, error info: %s." % (cmd, p.returncode, stderr))
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1493, in critical
    self._log(CRITICAL, msg, args, **kwargs)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1589, in _log
    self.handle(record)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1599, in handle
    self.callHandlers(record)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1661, in callHandlers
    hdlr.handle(record)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 952, in handle
    self.emit(record)
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/kit.py", line 42, in emit
    raise Exception(record.msg)
Exception: Command '/bin/sh path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split1/nextPolish.sh > path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split1/nextPolish.sh.o 2> path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split1/nextPolish.sh.e' returned non-zero exit status 127, error info: .
[183854 INFO] 2021-12-03 10:52:29 Submitted jobID:[183889] jobCmd:[path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split2/nextPolish.sh] in the local_cycle.
[183889 CRITICAL] 2021-12-03 10:52:29 Command '/bin/sh path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split2/nextPolish.sh > path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split2/nextPolish.sh.o 2> path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split2/nextPolish.sh.e' returned non-zero exit status 127, error info: .
Traceback (most recent call last):
  File "path/to/NextPolish.backup0/./nextPolish", line 515, in <module>
  File "path/to/NextPolish.backup0/./nextPolish", line 369, in main
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 347, in start
    self._start()
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 371, in _start
    self.submit(job)
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 255, in submit
    _, stdout, _ = self.run(job.cmd)
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 291, in run
    log.critical("Command '%s' returned non-zero exit status %d, error info: %s." % (cmd, p.returncode, stderr))
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1493, in critical
    self._log(CRITICAL, msg, args, **kwargs)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1589, in _log
    self.handle(record)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1599, in handle
    self.callHandlers(record)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1661, in callHandlers
    hdlr.handle(record)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 952, in handle
    self.emit(record)
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/kit.py", line 42, in emit
    raise Exception(record.msg)
Exception: Command '/bin/sh path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split2/nextPolish.sh > path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split2/nextPolish.sh.o 2> path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split2/nextPolish.sh.e' returned non-zero exit status 127, error info: .
[183854 INFO] 2021-12-03 10:52:30 Submitted jobID:[183895] jobCmd:[path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split3/nextPolish.sh] in the local_cycle.
[183895 CRITICAL] 2021-12-03 10:52:30 Command '/bin/sh path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split3/nextPolish.sh > path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split3/nextPolish.sh.o 2> path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split3/nextPolish.sh.e' returned non-zero exit status 127, error info: .
Traceback (most recent call last):
  File "path/to/NextPolish.backup0/./nextPolish", line 515, in <module>
  File "path/to/NextPolish.backup0/./nextPolish", line 369, in main
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 347, in start
    self._start()
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 371, in _start
    self.submit(job)
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 255, in submit
    _, stdout, _ = self.run(job.cmd)
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/task_control.py", line 291, in run
    log.critical("Command '%s' returned non-zero exit status %d, error info: %s." % (cmd, p.returncode, stderr))
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1493, in critical
    self._log(CRITICAL, msg, args, **kwargs)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1589, in _log
    self.handle(record)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1599, in handle
    self.callHandlers(record)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 1661, in callHandlers
    hdlr.handle(record)
  File "path/to/anaconda3/lib/python3.9/logging/__init__.py", line 952, in handle
    self.emit(record)
  File "path/to/anaconda3/lib/python3.9/site-packages/paralleltask/kit.py", line 42, in emit
    raise Exception(record.msg)
Exception: Command '/bin/sh path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split3/nextPolish.sh > path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split3/nextPolish.sh.o 2> path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split3/nextPolish.sh.e' returned non-zero exit status 127, error info: .
[183854 ERROR] 2021-12-03 10:52:37 db_split failed: please check the following logs:
[183854 ERROR] 2021-12-03 10:52:37 path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split1/nextPolish.sh.e
[183854 ERROR] 2021-12-03 10:52:37 path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split2/nextPolish.sh.e
[183854 ERROR] 2021-12-03 10:52:37 path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split3/nextPolish.sh.e

Regards, Alex

[moold]

Hi, could you paste the content of path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split1/nextPolish.sh.e to here?

[Gerlex89]

Hi,

path/to/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split1/nextPolish.sh.e:

hostname
+ hostname
cd path/to/NextPolish/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split1
+ cd path/to/NextPolish/NextPolish.backup0/00.score_chain/01.db_split.sh.work/db_split1
time path/to/NextPolish/NextPolish.backup0/bin/seq_split -d path/to/NextPolish/NextPolish.backup0 -m 315166.6666666667 -n 6 -t 5 -i 1 -s 1891000 -p input.sgspart path/to/NextPolish/NextPolish.backup0/test.fofn
+ time path/to/NextPolish/NextPolish.backup0/bin/seq_split -d path/to/NextPolish/NextPolish.backup0 -m 315166.6666666667 -n 6 -t 5 -i 1 -s 1891000 -p input.sgspart path/to/NextPolish/NextPolish.backup0/test.fofn
time: cannot run path/to/NextPolish/NextPolish.backup0/bin/seq_split: No such file or directory
Command exited with non-zero status 127
0.00user 0.00system 0:00.00elapsed ?%CPU (0avgtext+0avgdata 1020maxresident)k
0inputs+0outputs (0major+25minor)pagefaults 0swaps

[moold]

As the log says, there is no seq_split execution file, so follow here to reinstall. BTW, do not forget make command after downloading.

[Gerlex89]

I made the reinstall, but now I receive the log below from the nextPolish.sh.e file from running the test data. However, when I ran a clean installation of it in a server, then it's successful. Looks like is clear that the problem points to my local Python or Anaconda installation, but I would be glad if you have an idea what could be causing this issue. By now it's possible to close this question.

Thanks!

hostname
+ hostname
cd path/to/NextPolish/test_data/01_rundir/00.lgs_polish/04.polish.ref.sh.work/polish_genome1
+ cd path/to/NextPolish/test_data/01_rundir/00.lgs_polish/04.polish.ref.sh.work/polish_genome1
time /path/to/anaconda3/bin/python path/to/NextPolish/lib/nextpolish2.py -sp -p 1 -g path/to/NextPolish/test_data/./01_rundir/00.lgs_polish/input.genome.fasta -b path/to/NextPolish/test_data/./01_rundir/00.lgs_polish/input.genome.fasta.blc -i 0 -l path/to/NextPolish/test_data/./01_rundir/00.lgs_polish/lgs.sort.bam.list -r ont -o genome.nextpolish.part000.fasta
+ time /path/to/anaconda3/bin/python path/to/NextPolish/lib/nextpolish2.py -sp -p 1 -g path/to/NextPolish/test_data/./01_rundir/00.lgs_polish/input.genome.fasta -b path/to/NextPolish/test_data/./01_rundir/00.lgs_polish/input.genome.fasta.blc -i 0 -l path/to/NextPolish/test_data/./01_rundir/00.lgs_polish/lgs.sort.bam.list -r ont -o genome.nextpolish.part000.fasta
[110589 INFO] 2021-12-07 11:22:42 Corrected step options:
[110589 INFO] 2021-12-07 11:22:42 
split:                        0
process:                      1
auto:                         True
read_type:                    1
block_index:                  0
window:                       5000000
uppercase:                    False
alignment_score_ratio:        0.8
alignment_identity_ratio:     0.8
out:                          genome.nextpolish.part000.fasta
genome:                       path/to/NextPolish/test_data/./01_rundir/00.lgs_polish/input.genome.fasta
bam_list:                     path/to/NextPolish/test_data/./01_rundir/00.lgs_polish/lgs.sort.bam.list
block:                        path/to/NextPolish/test_data/./01_rundir/00.lgs_polish/input.genome.fasta.blc
[110589 WARNING] 2021-12-07 11:22:42 Adjust -p from 1 to 0, -w from 5000000 to 5000000, logical CPUs:4, available RAM:~6G, use -a to disable automatic adjustment.
Traceback (most recent call last):
  File "path/to/NextPolish/lib/nextpolish2.py", line 260, in <module>
    main(args)
  File "path/to/NextPolish/lib/nextpolish2.py", line 192, in main
    pool = Pool(args.process, initializer=start)
  File "/path/to/anaconda3/lib/python3.9/multiprocessing/context.py", line 119, in Pool
    return Pool(processes, initializer, initargs, maxtasksperchild,
  File "/path/to/anaconda3/lib/python3.9/multiprocessing/pool.py", line 205, in __init__
    raise ValueError("Number of processes must be at least 1")
ValueError: Number of processes must be at least 1
Command exited with non-zero status 1
0.08user 0.00system 0:00.09elapsed 100%CPU (0avgtext+0avgdata 16528maxresident)k
0inputs+8outputs (0major+2520minor)pagefaults 0swaps

[moold]

The RAM is too small

[Gerlex89]

Is there a proper way to increase it?

According to the FAQ of NextPolish it should be possible to increase it from the default 3G for Paralleltask, but I cannot see an effect in changing it in the cluster.cfg file or where precisely use this of the submit parameter. I still receive the same error.

Also how is that the memory isn't enough for only 3G?

[moold]

The computer node you submitted only have ~6Gb memory, you can not change it by adjusting parameters, you need to change another computer nodes to run it.

EDITED: Maybe you just forgot to change job_type = local to job_type = sge or others, if you want to submit your job on a computer cluster.

[Gerlex89]

Good to know. At least it's not a problem with my installation or files submitted.

These errors come from a local test. When the tool is used in a cluster it runs perfectly.

I was trying to figure out why there was such different behavior, but if it's due to a hardware restriction then there's nothing to do by now.

Thanks.