when i ran qtlseq on provided test dataset, i got below error.
bash qtlseq.sh
[QTL-seq:2025-12-09 20:30:28] Starting QTL-seq analysis.
[QTL-seq:2025-12-09 20:30:28] Indexing the reference FASTA.
[QTL-seq:2025-12-09 20:30:29] Reference FASTA indexing completed successfully.
[QTL-seq:2025-12-09 20:30:29] Aligning reads for parent.0000 using BWA.
[QTL-seq:2025-12-09 20:30:39] Alignment of parent.0000 completed successfully.
[QTL-seq:2025-12-09 20:30:29] Aligning reads for parent.0000 using BWA.
[QTL-seq:2025-12-09 20:30:39] Alignment of parent.0000 completed successfully.
[QTL-seq:2025-12-09 20:30:39] Aligning reads for bulk1.0000 using BWA.
[QTL-seq:2025-12-09 20:30:48] Alignment of bulk1.0000 completed successfully.
[QTL-seq:2025-12-09 20:30:48] Aligning reads for bulk2.0000 using BWA.
[QTL-seq:2025-12-09 20:30:58] Alignment of bulk2.0000 completed successfully.
[QTL-seq:2025-12-09 20:30:58] Sorting BAM files.
[QTL-seq:2025-12-09 20:31:37] BAM file sorting completed successfully.
[QTL-seq:2025-12-09 20:31:37] Calling variants.
[QTL-seq:2025-12-09 20:31:37] !!ERROR!! bcftools mpileup -a AD,ADF,ADR -B -q 40 -Q 18 -C 50 -O u -r test_chr -f test/10_ref/qtlseq_ref.fasta --ignore-RG test/20_bam/parent.bam test/20_bam/bulk1.bam test/20_bam/bulk2.bam 2>> test/log/bcftools.test_chr.log | bcftools call -vm -f GQ,GP -O u 2>> test/log/bcftools.test_chr.log | bcftools filter -i "INFO/MQ>=40" -O z -o test/30_vcf/qtlseq.test_chr.vcf.gz >> test/log/bcftools.test_chr.log 2>&1
please check below:
multiprocessing.pool.RemoteTraceback:
"""
Traceback (most recent call last):
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/mpileup.py", line 167, in mpileup
check=True)
File "/usr/lib64/python3.6/subprocess.py", line 438, in run
output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command 'bcftools mpileup -a AD,ADF,ADR -B -q 40 -Q 18 -C 50 -O u -r test_chr -f test/10_ref/qtlseq_ref.fasta --ignore-RG test/20_bam/parent.bam test/20_bam/bulk1.bam test/20_bam/bulk2.bam 2>> test/log/bcftools.test_chr.log | bcftools call -vm -f GQ,GP -O u 2>> test/log/bcftools.test_chr.log | bcftools filter -i "INFO/MQ>=40" -O z -o test/30_vcf/qtlseq.test_chr.vcf.gz >> test/log/bcftools.test_chr.log 2>&1' returned non-zero exit status 127.
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/usr/lib64/python3.6/multiprocessing/pool.py", line 119, in worker
result = (True, func(*args, **kwds))
File "/usr/lib64/python3.6/multiprocessing/pool.py", line 44, in mapstar
return list(map(*args))
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/mpileup.py", line 169, in mpileup
call_log(self.out, 'bcftools', cmd)
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/utils.py", line 20, in call_log
with open('{}/log/{}.log'.format(out_dir, name)) as log:
FileNotFoundError: [Errno 2] No such file or directory: 'test/log/bcftools.log'
"""
The above exception was the direct cause of the following exception:
Traceback (most recent call last):
File "/home/guest/.local/bin/qtlseq", line 11, in
load_entry_point('qtlseq==2.2.9', 'console_scripts', 'qtlseq')()
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/qtlseq.py", line 182, in main
QTLseq(args).run()
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/qtlseq.py", line 177, in run
self.mpileup()
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/qtlseq.py", line 132, in mpileup
mp.run()
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/mpileup.py", line 245, in run
p.map(self.mpileup, chr_names)
File "/usr/lib64/python3.6/multiprocessing/pool.py", line 266, in map
return self._map_async(func, iterable, mapstar, chunksize).get()
File "/usr/lib64/python3.6/multiprocessing/pool.py", line 644, in get
raise self._value
FileNotFoundError: [Errno 2] No such file or directory: 'test/log/bcftools.log'
when i ran qtlseq on provided test dataset, i got below error.
bash qtlseq.sh
[QTL-seq:2025-12-09 20:30:28] Starting QTL-seq analysis.
[QTL-seq:2025-12-09 20:30:28] Indexing the reference FASTA.
[QTL-seq:2025-12-09 20:30:29] Reference FASTA indexing completed successfully.
[QTL-seq:2025-12-09 20:30:29] Aligning reads for parent.0000 using BWA.
[QTL-seq:2025-12-09 20:30:39] Alignment of parent.0000 completed successfully.
[QTL-seq:2025-12-09 20:30:29] Aligning reads for parent.0000 using BWA.
[QTL-seq:2025-12-09 20:30:39] Alignment of parent.0000 completed successfully.
[QTL-seq:2025-12-09 20:30:39] Aligning reads for bulk1.0000 using BWA.
[QTL-seq:2025-12-09 20:30:48] Alignment of bulk1.0000 completed successfully.
[QTL-seq:2025-12-09 20:30:48] Aligning reads for bulk2.0000 using BWA.
[QTL-seq:2025-12-09 20:30:58] Alignment of bulk2.0000 completed successfully.
[QTL-seq:2025-12-09 20:30:58] Sorting BAM files.
[QTL-seq:2025-12-09 20:31:37] BAM file sorting completed successfully.
[QTL-seq:2025-12-09 20:31:37] Calling variants.
[QTL-seq:2025-12-09 20:31:37] !!ERROR!! bcftools mpileup -a AD,ADF,ADR -B -q 40 -Q 18 -C 50 -O u -r test_chr -f test/10_ref/qtlseq_ref.fasta --ignore-RG test/20_bam/parent.bam test/20_bam/bulk1.bam test/20_bam/bulk2.bam 2>> test/log/bcftools.test_chr.log | bcftools call -vm -f GQ,GP -O u 2>> test/log/bcftools.test_chr.log | bcftools filter -i "INFO/MQ>=40" -O z -o test/30_vcf/qtlseq.test_chr.vcf.gz >> test/log/bcftools.test_chr.log 2>&1
please check below:
multiprocessing.pool.RemoteTraceback:
"""
Traceback (most recent call last):
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/mpileup.py", line 167, in mpileup
check=True)
File "/usr/lib64/python3.6/subprocess.py", line 438, in run
output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command 'bcftools mpileup -a AD,ADF,ADR -B -q 40 -Q 18 -C 50 -O u -r test_chr -f test/10_ref/qtlseq_ref.fasta --ignore-RG test/20_bam/parent.bam test/20_bam/bulk1.bam test/20_bam/bulk2.bam 2>> test/log/bcftools.test_chr.log | bcftools call -vm -f GQ,GP -O u 2>> test/log/bcftools.test_chr.log | bcftools filter -i "INFO/MQ>=40" -O z -o test/30_vcf/qtlseq.test_chr.vcf.gz >> test/log/bcftools.test_chr.log 2>&1' returned non-zero exit status 127.
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/usr/lib64/python3.6/multiprocessing/pool.py", line 119, in worker
result = (True, func(*args, **kwds))
File "/usr/lib64/python3.6/multiprocessing/pool.py", line 44, in mapstar
return list(map(*args))
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/mpileup.py", line 169, in mpileup
call_log(self.out, 'bcftools', cmd)
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/utils.py", line 20, in call_log
with open('{}/log/{}.log'.format(out_dir, name)) as log:
FileNotFoundError: [Errno 2] No such file or directory: 'test/log/bcftools.log'
"""
The above exception was the direct cause of the following exception:
Traceback (most recent call last):
File "/home/guest/.local/bin/qtlseq", line 11, in
load_entry_point('qtlseq==2.2.9', 'console_scripts', 'qtlseq')()
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/qtlseq.py", line 182, in main
QTLseq(args).run()
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/qtlseq.py", line 177, in run
self.mpileup()
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/qtlseq.py", line 132, in mpileup
mp.run()
File "/home/guest/.local/lib/python3.6/site-packages/qtlseq/mpileup.py", line 245, in run
p.map(self.mpileup, chr_names)
File "/usr/lib64/python3.6/multiprocessing/pool.py", line 266, in map
return self._map_async(func, iterable, mapstar, chunksize).get()
File "/usr/lib64/python3.6/multiprocessing/pool.py", line 644, in get
raise self._value
FileNotFoundError: [Errno 2] No such file or directory: 'test/log/bcftools.log'