yusugihara / qtl-seq Goto Github PK

I came across an error，anyone got this problem too and how to slove it ? hope for you reply ，Thank you a lot .
!!ERROR!! tabix -f -p vcf qtl-seq_result2/30_vcf/qtlseq.Chr1.vcf.gz >> qtl-seq_result2/log/tabix.Chr1.log 2>&1

please check below:

[QTL-seq:2020-06-22 20:03:31] !!ERROR!! tabix -f -p vcf qtl-seq_result2/30_vcf/qtlseq.Chr2.vcf.gz >> qtl-seq_result2/log/tabix.Chr2.log 2>&1

please check below:

[QTL-seq:2020-06-22 20:07:10] !!ERROR!! tabix -f -p vcf qtl-seq_result2/30_vcf/qtlseq.Chr4.vcf.gz >> qtl-seq_result2/log/tabix.Chr4.log 2>&1

please check below:

Hi, I think it's a good package. But I had a problem. Because I use VCF in different formats, I can't use these packages. I only need to use these packages to calculate CI. Can you give me a method to calculate 99% and 95% CI only according to reads depth.

Hi!
May I have the full citation of Huang et al. (2019)?
I am curious about how the equation of the value 'k' was decided and affects the threshold.
Thank you very much!

Hi @YuSugihara
I came across this error, i couldn't figure out whether there is a problem while installing the package or some technical issue. Can you please help me out.

qtlseq -r R_genome.fasta -p QT4Rparent1.clean.fq.gz,QT4Rparent2.clean.fq.gz -b1 QT4Rpool1.clean.fq.gz,QT4Rpool2.clean.fq.gz -b2 QT4Spool1.clean.fq.gz,QT4Spool2.clean.fq.gz -n1 30 -n2 30 -o QT4new-dir -T
occur error as follows:
!!ERROR!! trimmomatic PE -threads 2 -phred33 QT4Rparent1.clean.fq.gz QT4Rparent2.clean.fq.gz QT4new-dir/00_fastq/parent.0000.1.trim.fastq.gz QT4new-dir/00_fastq/parent.0000.1.unpaired.fastq.gz QT4new-dir/00_fastq/parent.0000.2.trim.fastq.gz QT4new-dir/00_fastq/parent.0000.2.unpaired.fastq.gz LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:75 >> QT4new-dir/log/trimmomatic.log 2>&1

Hello! I am having problems with generating my QTLplot.
 
I have run QTLseq for my sequences and the initial QTLplot from this run has does not have a smooth red line. So I decided to run QTLplot using the vcf file from my QTLseq run using the code below:
 
qtlplot -v qtlseq.vcf.gz
        -o bsa_plot
        -n1 10
        -n2 10
        -w 2000
        -s 100

Here’s the picture:

I tried changing the -w to 1000 and the chromosomes are okay. But running the same -w 1000 for the second time, chromosomes 1-5 are lost (picture below). Same with when I run -w 3000, first one is okay and the rerun has missing chromosomes.

-w 1000 rerun

-w 3000 rerun

I don't understand why. Hope you could help. Thank you very much! :)

hello I have a question about some error

I downloaded the test file and ran it in the way of example 2.

qtlseq -r reference.fasta
-p parent.1.fastq,parent.2.fastq
-b1 bulk_1.1.fastq,bulk_1.2.fastq
-b2 bulk_2.1.fastq,bulk_2.2.fastq
-n1 20
-n2 20
-o example_dir
-T

[QTL-seq:2023-09-18 15:04:12] start to run QTL-seq.
[QTL-seq:2023-09-18 15:04:12] maximum number of threads which you can use is up to 8.
Traceback (most recent call last):
File "/home/jjw/miniconda3/envs/qtl/bin/qtlseq", line 33, in
sys.exit(load_entry_point('qtlseq', 'console_scripts', 'qtlseq')())
File "/home/jjw/QTL-seq/qtlseq/qtlseq.py", line 173, in main
QTLseq(args).run()
File "/home/jjw/QTL-seq/qtlseq/qtlseq.py", line 36, in init
self.link_ref()
File "/home/jjw/QTL-seq/qtlseq/qtlseq.py", line 63, in link_ref
os.symlink(path_to_ref, sym_ref)
OSError: [Errno 38] Function not implemented: '/media/jjw/seagate/hy/fasta/qtlseq_ref.fasta' -> 'test/10_ref/qtlseq_ref.fasta'

However, an error occurred. The error contents are as follows.

OSError: [Errno 38] Function not implemented: 'qtlseq_ref.fasta' -> 'example/10_ref/qtlseq_ref.fasta'

Please tell me the solution or the cause of the problem

Hi,

I am running QTLseq from bams and I get the following error with the following code.

The bams have all been mapped with bwa mem then run through the following picard modules CleanSam, FixMateInformation, AddOrReplaceReadGroups, and MarkDuplicatesWithMateCigar

qtlseq -r refrence.fasta  -p parent_cleaned_fixed_group_DEDUP.bam -b1 ../Bulk1_cleaned_fixed_group_DEDUP.bam -b2 ../Bulk2_cleaned_fixed_group_DEDUP.bam -n1 38 -n2 38  -o ../QTLseq_output
[QTL-seq:2021-04-25 15:02:54] start to run QTL-seq.
[QTL-seq:2021-04-25 15:02:54] maximum number of threads which you can use is up to 32.
[QTL-seq:2021-04-25 15:02:54] start to index reference fasta.
[QTL-seq:2021-04-25 15:18:39] indexing of reference successfully finished.
[QTL-seq:2021-04-25 15:18:39] start to merge BAMs.
[QTL-seq:2021-04-25 19:37:00] merging process successfully finished.
[QTL-seq:2021-04-25 19:37:00] start to call variants.
[QTL-seq:2021-04-25 20:00:14] variant calling successfully finished.
[QTL-seq:2021-04-25 20:00:14] start to index VCF.
[QTL-seq:2021-04-25 20:00:15] indexing VCF successfully finished.
[QTL-seq:2021-04-25 20:00:15] start to run QTL-plot.
[QTL-seq:2021-04-25 20:00:15] maximum number of threads which you can use is up to 32.
[QTL-seq:2021-04-25 20:00:15] start to calculate SNP-index.
Traceback (most recent call last):
  File "/home/cgc-genomics-admin/miniconda3/envs/BULK_QTL_env/bin/qtlplot", line 10, in <module>
    sys.exit(main())
  File "/home/cgc-genomics-admin/miniconda3/envs/BULK_QTL_env/lib/python3.9/site-packages/qtlseq/qtlplot.py", line 207, in main
    qp.run()
  File "/home/cgc-genomics-admin/miniconda3/envs/BULK_QTL_env/lib/python3.9/site-packages/qtlseq/qtlplot.py", line 192, in run
    v2i.run()
  File "/home/cgc-genomics-admin/miniconda3/envs/BULK_QTL_env/lib/python3.9/site-packages/qtlseq/vcf2index.py", line 342, in run
    self.field_pos = self.get_field()
  File "/home/cgc-genomics-admin/miniconda3/envs/BULK_QTL_env/lib/python3.9/site-packages/qtlseq/vcf2index.py", line 130, in get_field
    return GT_pos, AD_pos, ADF_pos, ADR_pos
UnboundLocalError: local variable 'GT_pos' referenced before assignment
[QTL-seq:2021-04-25 20:00:15] QTL-seq successfully finished.

Hi,
I got an error when using QTL-seq.It said "FileNotFoundError: [Errno 2] No such file or directory: 'QTLseq/snp_index.tsv.temp'",but the temp shouldn't be created by the software?I don't know why.

Below is Traceback and error
Traceback (most recent call last): File "/home/zwang/anaconda3/envs/SNP/bin/qtlplot", line 10, in <module> sys.exit(main()) File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/qtlseq/qtlplot.py", line 208, in main qp.run() File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/qtlseq/qtlplot.py", line 193, in run v2i.run() File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/qtlseq/vcf2index.py", line 343, in run self.calculate_SNPindex() File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/qtlseq/vcf2index.py", line 338, in calculate_SNPindex self.table_sort() File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/qtlseq/vcf2index.py", line 297, in table_sort 'delta_SNPindex']) File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/pandas/io/parsers.py", line 610, in read_csv return _read(filepath_or_buffer, kwds) File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/pandas/io/parsers.py", line 462, in _read parser = TextFileReader(filepath_or_buffer, **kwds) File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/pandas/io/parsers.py", line 819, in __init__ self._engine = self._make_engine(self.engine) File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/pandas/io/parsers.py", line 1050, in _make_engine return mapping[engine](self.f, **self.options) # type: ignore[call-arg] File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/pandas/io/parsers.py", line 1867, in __init__ self._open_handles(src, kwds) File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/pandas/io/parsers.py", line 1368, in _open_handles storage_options=kwds.get("storage_options", None), File "/home/zwang/anaconda3/envs/SNP/lib/python3.7/site-packages/pandas/io/common.py", line 647, in get_handle newline="", FileNotFoundError: [Errno 2] No such file or directory: 'QTLseq/snp_index.tsv.temp'

My command is
qtlplot -v ./ZM895_Rbuk_Sbuk_merged.vcf.gz \ -o QTLseq \ -n1 30 \ -n2 30 \ -F 2 \ --species Wheat \ -w 2000 \ -s 200

The vcf file "ZM895_Rbuk_Sbuk_merged.vcf.gz" contains AD format and three columns of parent, bulk1 and bulk2 in this order.And I have created the output file folder QTLseq.

The vcf file is like
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT ZM895_FRAS220033950-2r ZM-Rbuk_FRAS220033948-2r ZM-Sbuk_FRAS220033949-2r 1A 7638 . A C 213.78 . BaseQRankSum=2.638;ClippingRankSum=-0;ExcessHet=3.0103;FS=0;MQ=60;MQRankSum=-0;QD=21.38;ReadPosRankSum=0.61;SOR=0.569;DP=10;AF=0.5;MLEAC=1;MLEAF=0.5;AN=2;AC=1 GT:AD:DP:GQ:PL ./.:.:.:.:. 0/1:3,7:10:25:242,0,25 ./.:.:.:.:.

Thanks!

I was trying with test data as you provided. I got an error and solved it.

Dear doctor:
Thanks for your outstanding for this QTL-seq tool. But some question appear when I use the qtlseq software. When I use the test data to perform below code:

qtlseq -o test -n1 20 -n2 20  -w 100 -s 20 -r qtlseq_ref.fasta -p qtlseq_parent.1.fastq.gz,qtlseq_parent.2.fastq.gz \
 -b1 qtlseq_bulk1.1.fastq.gz,qtlseq_bulk1.2.fastq.gz \
 -b2 qtlseq_bulk2.1.fastq.gz,qtlseq_bulk2.2.fastq.gz

And then, there are not warring and error happing, like this

[QTL-seq:2022-01-11 11:33:45] start to run QTL-seq.
[QTL-seq:2022-01-11 11:33:45] maximum number of threads which you can use is up to 160.
[QTL-seq:2022-01-11 11:33:45] start to index reference fasta.
[QTL-seq:2022-01-11 11:33:46] indexing of reference successfully finished.
[QTL-seq:2022-01-11 11:33:46] start to align reads of parent.0000 by BWA.
[QTL-seq:2022-01-11 11:33:52] alignment parent.0000 successfully finished.
[QTL-seq:2022-01-11 11:33:52] start to align reads of bulk1.0000 by BWA.
[QTL-seq:2022-01-11 11:33:58] alignment bulk1.0000 successfully finished.
[QTL-seq:2022-01-11 11:33:58] start to align reads of bulk2.0000 by BWA.
[QTL-seq:2022-01-11 11:34:04] alignment bulk2.0000 successfully finished.
[QTL-seq:2022-01-11 11:34:04] start to merge BAMs.
[QTL-seq:2022-01-11 11:34:06] merging process successfully finished.
[QTL-seq:2022-01-11 11:34:06] start to call variants.
[QTL-seq:2022-01-11 11:34:31] variant calling successfully finished.
[QTL-seq:2022-01-11 11:34:31] start to index VCF.
[QTL-seq:2022-01-11 11:34:31] indexing VCF successfully finished.
[QTL-seq:2022-01-11 11:34:32] start to run QTL-plot.
[QTL-seq:2022-01-11 11:34:32] maximum number of threads which you can use is up to 160.
[QTL-seq:2022-01-11 11:34:32] start to calculate SNP-index.
[QTL-seq:2022-01-11 11:34:53] SNP-index successfully finished.
[QTL-seq:2022-01-11 11:34:53] start to smooth SNP-index.
[QTL-seq:2022-01-11 11:34:53] smoothing process successfully finished.
[QTL-seq:2022-01-11 11:34:53] plotting now...
[QTL-seq:2022-01-11 11:34:54] QTL-plot successfully finished.
[QTL-seq:2022-01-11 11:34:54] QTL-seq successfully finished.

However, When I check the figure in 40_qtlseq file, there are no smooth lines in "bulk1_SNPindex.png,bulk2_SNPindex.png and delta_SNPindex.png", as the example follow show:

Could you give some advice for that? Thanks again for your help!

Best regards,
Gaoxiang Ji

How to use this with two parent and two bulk?
Thanks you for your code supply for us!
The guide tell us just use a single whole-genome resequencing of either of the parental cultivars. if so, whether the accury of result maybe lower than use two parent?
If use two parents, how can I use your code?

Please help with this error. I'm using version 2.2.3 installed with conda. In fact, I am providing sorted bams and can skip this step.

qtlseq -r normalised_deepti_sm.fasta -p a93.our_sm.sorted.rmdup.bam -b1 F1.our_sm.sorted.rmdup.bam -b2 F6.our_sm.sorted.rmdup.bam -n1 8 -n2 8 -o qtlseq_output
[QTL-seq:2022-06-24 16:37:19] start to run QTL-seq.
[QTL-seq:2022-06-24 16:37:19] maximum number of threads which you can use is up to 16.
[QTL-seq:2022-06-24 16:37:19] start to index reference fasta.
[QTL-seq:2022-06-24 16:42:45] indexing of reference successfully finished.
[QTL-seq:2022-06-24 16:42:45] start to merge BAMs.
[QTL-seq:2022-06-24 16:42:45] !!ERROR!! samtools sort -m 1G -@ 2 -o qtlseq_output/20_bam/parent.bam qtlseq_output/20_bam/parent.unsorted.bam >> qtlseq_output/log/samtools.log 2>&1

please check below:

open: No such file or directory

Dear developer,

The Samtools error information as following stops the pipeline. I think it's not really an error.

[QTL-seq:2022-12-06 10:14:05] start to merge BAMs.
[QTL-seq:2022-12-06 10:52:35] !!ERROR!! samtools sort -m 1G -@ 40 -o 008qtlseq/qtlseq_out/20_bam/bulk1.bam 008qtlseq/qtlseq_out/20_bam/bulk1.unsorted.bam >> 008qtlseq/qtlseq_out/log/samtools.log 2>&1

It's giving notification.
[W::bam_merge_core2] No @hd tag found error.
Program terminated after generating BAM file.
I enclosed the log file.
bwa.log
trimmomatic.log
samtools.log

Hello, I am trying to perform QTL-seq using the test data (https://github.com/YuSugihara/QTL-seq/tree/master/test) in the miniconda virtual environment.
But the process does not start and encounters the error messages below.
Could you give some suggestions? Thanks in advance.

(qtlseq) yoshio@iMac 210209_test % ./qtlseq.sh
Traceback (most recent call last):
  File "<string>", line 1, in <module>
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/spawn.py", line 116, in spawn_main
    exitcode = _main(fd, parent_sentinel)
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/spawn.py", line 125, in _main
    prepare(preparation_data)
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/spawn.py", line 236, in prepare
    _fixup_main_from_path(data['init_main_from_path'])
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/spawn.py", line 287, in _fixup_main_from_path
    main_content = runpy.run_path(main_path,
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/runpy.py", line 268, in run_path
    return _run_module_code(code, init_globals, run_name,
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/runpy.py", line 97, in _run_module_code
    _run_code(code, mod_globals, init_globals,
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/runpy.py", line 87, in _run_code
    exec(code, run_globals)
  File "/Users/yoshio/miniconda3/envs/qtlseq/bin/qtlseq", line 7, in <module>
    from qtlseq.qtlseq import main
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/site-packages/qtlseq/qtlseq.py", line 18, in <module>
    from qtlseq.mpileup import Mpileup
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/site-packages/qtlseq/mpileup.py", line 7, in <module>
    from qtlseq.vcf2index import Vcf2Index
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/site-packages/qtlseq/vcf2index.py", line 10, in <module>
    cache = Manager().dict()
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/context.py", line 57, in Manager
    m.start()
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/managers.py", line 553, in start
    self._process.start()
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/process.py", line 121, in start
    self._popen = self._Popen(self)
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/context.py", line 284, in _Popen
    return Popen(process_obj)
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/popen_spawn_posix.py", line 32, in __init__
    super().__init__(process_obj)
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/popen_fork.py", line 19, in __init__
    self._launch(process_obj)
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/popen_spawn_posix.py", line 42, in _launch
    prep_data = spawn.get_preparation_data(process_obj._name)
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/spawn.py", line 154, in get_preparation_data
    _check_not_importing_main()
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/spawn.py", line 134, in _check_not_importing_main
    raise RuntimeError('''
RuntimeError: 
        An attempt has been made to start a new process before the
        current process has finished its bootstrapping phase.

        This probably means that you are not using fork to start your
        child processes and you have forgotten to use the proper idiom
        in the main module:

            if __name__ == '__main__':
                freeze_support()
                ...

        The "freeze_support()" line can be omitted if the program
        is not going to be frozen to produce an executable.
Traceback (most recent call last):
  File "/Users/yoshio/miniconda3/envs/qtlseq/bin/qtlseq", line 7, in <module>
    from qtlseq.qtlseq import main
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/site-packages/qtlseq/qtlseq.py", line 18, in <module>
    from qtlseq.mpileup import Mpileup
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/site-packages/qtlseq/mpileup.py", line 7, in <module>
    from qtlseq.vcf2index import Vcf2Index
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/site-packages/qtlseq/vcf2index.py", line 10, in <module>
    cache = Manager().dict()
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/context.py", line 57, in Manager
    m.start()
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/managers.py", line 557, in start
    self._address = reader.recv()
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/connection.py", line 255, in recv
    buf = self._recv_bytes()
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/connection.py", line 419, in _recv_bytes
    buf = self._recv(4)
  File "/Users/yoshio/miniconda3/envs/qtlseq/lib/python3.9/multiprocessing/connection.py", line 388, in _recv
    raise EOFError
EOFError

Hello
I am working on the wheat genome and the pipeline crashes because the chromosomes are too big for the .bai index. Can you please suggest a fix with the .csi index?

Hi,
Many thanks to the developers.
I used use the mutmap v2.3.3 and showed "MutMap successfully finished". But the message "SystemError: tile cannot extend outside image" showed in the last line. I don't know how to deal with it.

Could you help me?

Best wishes!

Hi,
I'm working with QTL-seq pipeline and have successfully finished the analysis using parent 1. However, when I tried to redo the analysis using parent 2, it always stopped in the delta SNP calculation step. After I investigate, there might be an issue with BAM file for parent 2 which is too small and not reasonable. I have tried to use a new copy of the fastq file of parent 2 but still results in the same error.
Could anyone help me with this?

Thanks!

Hello,
I am stuck at the part where snpEff has to be used because the snpEff version downloaded along with qtlseq is version 5.12, made with java 12. The conda does not have java 12 package yet. How can I use snpEff for qtlseq then? Please help me.

I got an error like this and it stopped.

[QTL-seq:2023-07-17 23:05:13] start to run QTL-seq.
[QTL-seq:2023-07-17 23:05:13] maximum number of threads which you can use is up to 16.
[QTL-seq:2023-07-17 23:05:13] start to index reference fasta.
[QTL-seq:2023-07-17 23:05:14] indexing of reference successfully finished.
[QTL-seq:2023-07-17 23:05:14] start to align reads of parent.0000 by BWA.
[QTL-seq:2023-07-17 23:05:19] alignment parent.0000 successfully finished.
[QTL-seq:2023-07-17 23:05:19] start to align reads of bulk1.0000 by BWA.
[QTL-seq:2023-07-17 23:05:23] alignment bulk1.0000 successfully finished.
[QTL-seq:2023-07-17 23:05:23] start to align reads of bulk2.0000 by BWA.
[QTL-seq:2023-07-17 23:05:28] alignment bulk2.0000 successfully finished.
[QTL-seq:2023-07-17 23:05:28] start to merge BAMs.
[QTL-seq:2023-07-17 23:05:30] merging process successfully finished.
[QTL-seq:2023-07-17 23:05:30] start to call variants.
Process SpawnPoolWorker-2:
Traceback (most recent call last):
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/process.py", line 315, in _bootstrap
self.run()
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/process.py", line 108, in run
self._target(*self._args, **self._kwargs)
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/pool.py", line 114, in worker
task = get()
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/queues.py", line 367, in get
return _ForkingPickler.loads(res)
File "/Users/jiuhaizhao/Documents/QTLseq/QTL-seq/qtlseq/mpileup.py", line 7, in
from qtlseq.vcf2index import Vcf2Index
File "/Users/jiuhaizhao/Documents/QTLseq/QTL-seq/qtlseq/vcf2index.py", line 10, in
cache = Manager().dict()
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/context.py", line 57, in Manager
m.start()
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/managers.py", line 554, in start
self._process.start()
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/process.py", line 118, in start
assert not _current_process._config.get('daemon'),
AssertionError: daemonic processes are not allowed to have children
^CProcess SpawnPoolWorker-4:
Process SpawnPoolWorker-3:
Traceback (most recent call last):
File "/Users/jiuhaizhao/miniconda3/bin/qtlseq", line 33, in
sys.exit(load_entry_point('qtlseq', 'console_scripts', 'qtlseq')())
File "/Users/jiuhaizhao/Documents/QTLseq/QTL-seq/qtlseq/qtlseq.py", line 171, in main
Traceback (most recent call last):
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/process.py", line 315, in _bootstrap
self.run()
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/process.py", line 108, in run
self._target(*self._args, **self._kwargs)
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/pool.py", line 114, in worker
task = get()
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/queues.py", line 364, in get
with self._rlock:
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/synchronize.py", line 95, in enter
return self._semlock.enter()
Traceback (most recent call last):
KeyboardInterrupt
QTLseq(args).run()
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/process.py", line 315, in _bootstrap
self.run()
File "/Users/jiuhaizhao/Documents/QTLseq/QTL-seq/qtlseq/qtlseq.py", line 166, in run
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/process.py", line 108, in run
self._target(*self._args, **self._kwargs)
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/pool.py", line 114, in worker
task = get()
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/queues.py", line 365, in get
res = self._reader.recv_bytes()
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/connection.py", line 221, in recv_bytes
buf = self._recv_bytes(maxlength)
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/connection.py", line 419, in _recv_bytes
buf = self._recv(4)
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/connection.py", line 384, in _recv
chunk = read(handle, remaining)
KeyboardInterrupt
self.mpileup()
File "/Users/jiuhaizhao/Documents/QTLseq/QTL-seq/qtlseq/qtlseq.py", line 122, in mpileup
mp.run()
File "/Users/jiuhaizhao/Documents/QTLseq/QTL-seq/qtlseq/mpileup.py", line 228, in run
p.map(self.mpileup, chr_names)
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/pool.py", line 364, in map
return self._map_async(func, iterable, mapstar, chunksize).get()
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/pool.py", line 765, in get
self.wait(timeout)
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/multiprocessing/pool.py", line 762, in wait
self._event.wait(timeout)
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/threading.py", line 581, in wait
signaled = self._cond.wait(timeout)
File "/Users/jiuhaizhao/miniconda3/lib/python3.9/threading.py", line 312, in wait
waiter.acquire()
KeyboardInterrupt

Hi,

It seems that this pipeline can only use for RIL or F2 population, why not make some modifies to accept BC populaiton?

Best,
Kun

Hi sir,
I have run qtlseq with vcf file and getting error related to image size. I am providing the errors observed during the run for your intervention.
Help me to solve this.

[QTL-seq:2021-06-23 22:04:16] start to run QTL-plot.
[QTL-seq:2021-06-23 22:04:16] maximum number of threads which you can use is up to 40.
[QTL-seq:2021-06-23 22:04:16] start to calculate SNP-index.
[QTL-seq:2021-06-23 22:04:17] no ADF or ADR field in your VCF.
[QTL-seq:2021-06-23 22:04:17] strand bias filter will be skipped.
[QTL-seq:2021-06-23 22:19:18] SNP-index successfully finished.
[QTL-seq:2021-06-23 22:19:18] start to smooth SNP-index.
[QTL-seq:2021-06-23 22:19:24] smoothing process successfully finished.
[QTL-seq:2021-06-23 22:19:24] plotting now...
Traceback (most recent call last):
File "/home/iipruser/yes/bin/qtlplot", line 10, in
sys.exit(main())
File "/home/iipruser/yes/lib/python3.8/site-packages/qtlseq/qtlplot.py", line 208, in main
qp.run()
File "/home/iipruser/yes/lib/python3.8/site-packages/qtlseq/qtlplot.py", line 197, in run
pt.run()
File "/home/iipruser/yes/lib/python3.8/site-packages/qtlseq/plot.py", line 268, in run
self.plot_bulk1_SNPindex()
File "/home/iipruser/yes/lib/python3.8/site-packages/qtlseq/plot.py", line 140, in plot_bulk1_SNPindex
plt.savefig('{}/bulk1_SNPindex.{}'.format(self.out, self.args.format))
File "/home/iipruser/yes/lib/python3.8/site-packages/matplotlib/pyplot.py", line 859, in savefig
res = fig.savefig(*args, **kwargs)
File "/home/iipruser/yes/lib/python3.8/site-packages/matplotlib/figure.py", line 2311, in savefig
self.canvas.print_figure(fname, **kwargs)
File "/home/iipruser/yes/lib/python3.8/site-packages/matplotlib/backend_bases.py", line 2210, in print_figure
result = print_method(
File "/home/iipruser/yes/lib/python3.8/site-packages/matplotlib/backend_bases.py", line 1639, in wrapper
return func(*args, **kwargs)
File "/home/iipruser/yes/lib/python3.8/site-packages/matplotlib/backends/backend_agg.py", line 509, in print_png
FigureCanvasAgg.draw(self)
File "/home/iipruser/yes/lib/python3.8/site-packages/matplotlib/backends/backend_agg.py", line 402, in draw
self.renderer = self.get_renderer(cleared=True)
File "/home/iipruser/yes/lib/python3.8/site-packages/matplotlib/backends/backend_agg.py", line 418, in get_renderer
self.renderer = RendererAgg(w, h, self.figure.dpi)
File "/home/iipruser/yes/lib/python3.8/site-packages/matplotlib/backends/backend_agg.py", line 96, in init
self._renderer = _RendererAgg(int(width), int(height), dpi)
ValueError: Image size of 1500x1245600 pixels is too large. It must be less than 2^16 in each direction.

Hi,

Can I run your pipeline without parent sample? Or why parent is a necessary conditions?

Best wishes,
Kun

Hi,

I am trying to run your pipeline from bam files.

I use the launch command:

qtlseq -r ref.fasta \
       -t 30 \
      -p  /20_bam/parent.bam \
      -b1 /20_bam/bulk1.bam \
      -b2 /20_bam/bulk2.bam \
      -n1 38 \
      -n2 38 \
      -o ../out

However I get the following error:

QTL-seq:2021-05-03 11:36:27] start to run QTL-seq.
[QTL-seq:2021-05-03 11:36:27] maximum number of threads which you can use is up to 32.
[QTL-seq:2021-05-03 11:36:27] start to index reference fasta.
[QTL-seq:2021-05-03 11:52:37] indexing of reference successfully finished.
[QTL-seq:2021-05-03 11:52:37] start to merge BAMs.
[QTL-seq:2021-05-03 11:52:37] !!ERROR!! samtools sort -m 1G -@ 30 -o ../out/20_bam/parent.bam 
../out/20_bam/parent.unsorted.bam >> 
../out/log/samtools.log 2>&1

please check below:
[E::hts_open_format] Failed to open file "
../out/20_bam/parent.unsorted.bam" : No such file or directory
samtools sort: can't open "../out/20_bam/parent.unsorted.bam": No such file or directory

Is parent.unsorted.bam something I was supposed to generate?

After running the code

qtlseq --ref GCF_000741045.1_Vradiata_ver6_genomic.fna --parent parent.0000.1.trim.fastq,parent.0000.2.trim.fastq --bulk1 output_R1_paired.fq,output_R2_paired.fq --bulk2 output_S1_paired.fq,output_S2_paired.fq --N-bulk1 20 --N-bulk2 20 --out results1 --filial 8 --threads 20

[QTL-seq:2023-03-02 21:25:16] start to run QTL-seq.
[QTL-seq:2023-03-02 21:25:16] maximum number of threads which you can use is up to 20.
!!WARNING!! You can use up to 20 threads. This program will use 20 threads.
[QTL-seq:2023-03-02 21:25:16] start to index reference fasta.
[QTL-seq:2023-03-02 21:32:59] indexing of reference successfully finished.
[QTL-seq:2023-03-02 21:32:59] start to align reads of parent.0000 by BWA.

[QTL-seq:2023-03-02 21:34:03] !!ERROR!! bwa mem -t 20 results1/10_ref/GCF_000741045.1_Vradiata_ver6_genomic.fna parent.0000.1.trim.fastq parent.0000.2.trim.fastq | samtools fixmate -m - - | samtools sort -m 1G -@ 20 | samtools markdup -r - - | samtools view -b -f 2 -F 2048 -o results1/20_bam/parent.0000.bam >> results1/log/alignment.log 2>&1

please check below:

[main_samview] fail to read the header from "-".

Hi, @YuSugihara!

I would like to be clarified on which sequence to input for the --parent option in QTL-seq - Is it the parent that shows the desired phenotype or the one that does not?

Similarly, what should be the input for --cultivar in MutMap: wildtype or the mutant sequence? Your reply to these would be of great help.

Best!
Gokulan.

Hi, I am getting the following error in QTLseq pipeline, Please help to resolve

[QTL-seq:2020-12-17 14:11:25] indexing VCF successfully finished.
Traceback (most recent call last):
File "/home/haritha/.local/bin/qtlplot", line 11, in
load_entry_point('qtlseq', 'console_scripts', 'qtlplot')()
File "/home/haritha/.local/lib/python2.7/site-packages/pkg_resources/init.py", line 489, in load_entry_point
return get_distribution(dist).load_entry_point(group, name)
File "/home/haritha/.local/lib/python2.7/site-packages/pkg_resources/init.py", line 2852, in load_entry_point
return ep.load()
File "/home/haritha/.local/lib/python2.7/site-packages/pkg_resources/init.py", line 2443, in load
return self.resolve()
File "/home/haritha/.local/lib/python2.7/site-packages/pkg_resources/init.py", line 2449, in resolve
module = import(self.module_name, fromlist=['name'], level=0)
File "/home/haritha/QTL-seq/qtlseq/qtlplot.py", line 31
flush=True)
^
SyntaxError: invalid syntax
[QTL-seq:2020-12-17 14:11:26] QTL-seq successfully finished.

Hello.
I'm new to bioinformatics and I have some questions regarding QTL-seq as I'm experimenting this method.

I detected some INDELS with delta-SNP-index of 1. However, as I try to visualize them using IGV, they seem to be off-target (usually some bases to the right). Here in the picture I have an INDEL that was supposed to be ..67-..68, yet the deletion seems to be in ..69 position. Furthermore, bases in the parent and the two progeny bulks are identical, which was not what I expected (accordingly, the low phenotype should contain only A but most of their reads still have G after that).

Did I do anything incorrectly?

Hello, what would be the filial generation for pseudo-test cross?

With best regards

Sandip

Hi.

The output of my run results in a single file with many plots with the same x-axis. I would like to use the raw data to plot myself using ggplot2 so that I can manipulate the axis, highlight datapoints, etc. How can I obtain the raw data that was used to generate the outputs in directory 40_qtlseq?

Thanks!

I used it well before, but this time I don’t know what happened.
the version is qtlseq 2.1.2。
[QTL-seq:2020-09-08 00:03:05] [QTL-seq:2020-09-08 00:03:05] !!ERROR!! bcftools mpileup -a AD,ADF,ADR -B -q 40 -Q 18 -C 50 -O u -r chr03 -f stigma3/10_ref/IRGSP-1.0_genome.fasta --ignore-RG stigma3/20_bam/parent.filt.bam stigma3/20_bam/bulk1.filt.bam stigma3/20_bam/bulk2.filt.bam | bcftools call -vm -f GQ,GP -O u | bcftools filter -i "INFO/MQ>=40" -O z -o stigma3/30_vcf/qtlseq.chr03.vcf.gz >> stigma3/log/bcftools.chr03.log 2>&1
please check below:

please check below:

multiprocessing.pool.RemoteTraceback:
"""
Traceback (most recent call last):
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/mpileup.py", line 144, in mpileup
check=True)
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/subprocess.py", line 512, 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 chr09 -f stigma3/10_ref/IRGSP-1.0_genome.fasta --ignore-RG stigma3/20_bam/parent.filt.bam stigma3/20_bam/bulk1.filt.bam stigma3/20_bam/bulk2.filt.bam | bcftools call -vm -f GQ,GP -O u | bcftools filter -i "INFO/MQ>=40" -O z -o stigma3/30_vcf/qtlseq.chr09.vcf.gz >> stigma3/log/bcftools.chr09.log 2>&1' returned non-zero exit status 255.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/multiprocessing/pool.py", line 121, in worker
result = (True, func(*args, **kwds))
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/multiprocessing/pool.py", line 44, in mapstar
return list(map(*args))
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/mpileup.py", line 146, in mpileup
call_log(self.out, 'bcftools', cmd1)
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/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: 'stigma3/log/bcftools.log'
"""

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
File "/home/xiehan1223/miniconda3/envs/bioinfo/bin/qtlseq", line 10, in
sys.exit(main())
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/qtlseq.py", line 173, in main
QTLseq(args).run()
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/qtlseq.py", line 168, in run
self.mpileup()
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/qtlseq.py", line 123, in mpileup
mp.run()
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/mpileup.py", line 229, in run
p.map(self.mpileup, chr_names)
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/multiprocessing/pool.py", line 268, in map
return self._map_async(func, iterable, mapstar, chunksize).get()
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/multiprocessing/pool.py", line 657, in get
raise self._value
FileNotFoundError: [Errno 2] No such file or directory: 'stigma3/log/bcftools.log'stigma3/20_bam/parent.filt.bam stigma3/20_bam/bulk1.filt.bam stigma3/20_bam/bulk2.filt.bam | bcftools call -vm -f GQ,GP -O u | bcftools filter -i "INFO/MQ>=40" -O z -o stigma3/30_vcf/qtlseq.chr03.vcf.gz >> stigma3/log/bcftools.chr03.log 2>&1
please check below:

please check below:

multiprocessing.pool.RemoteTraceback:
"""
Traceback (most recent call last):
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/mpileup.py", line 144, in mpileup
check=True)
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/subprocess.py", line 512, 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 chr09 -f stigma3/10_ref/IRGSP-1.0_genome.fasta --ignore-RG stigma3/20_bam/parent.filt.bam stigma3/20_bam/bulk1.filt.bam stigma3/20_bam/bulk2.filt.bam | bcftools call -vm -f GQ,GP -O u | bcftools filter -i "INFO/MQ>=40" -O z -o stigma3/30_vcf/qtlseq.chr09.vcf.gz >> stigma3/log/bcftools.chr09.log 2>&1' returned non-zero exit status 255.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/multiprocessing/pool.py", line 121, in worker
result = (True, func(*args, **kwds))
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/multiprocessing/pool.py", line 44, in mapstar
return list(map(*args))
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/mpileup.py", line 146, in mpileup
call_log(self.out, 'bcftools', cmd1)
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/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: 'stigma3/log/bcftools.log'
"""

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
File "/home/xiehan1223/miniconda3/envs/bioinfo/bin/qtlseq", line 10, in
sys.exit(main())
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/qtlseq.py", line 173, in main
QTLseq(args).run()
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/qtlseq.py", line 168, in run
self.mpileup()
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/qtlseq.py", line 123, in mpileup
mp.run()
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/site-packages/qtlseq/mpileup.py", line 229, in run
p.map(self.mpileup, chr_names)
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/multiprocessing/pool.py", line 268, in map
return self._map_async(func, iterable, mapstar, chunksize).get()
File "/home/xiehan1223/miniconda3/envs/bioinfo/lib/python3.7/multiprocessing/pool.py", line 657, in get
raise self._value
FileNotFoundError: [Errno 2] No such file or directory: 'stigma3/log/bcftools.log'

please suggest, how to rectify this error.

[QTL-seq:2024-02-09 21:00:33] !!ERROR!! bcftools mpileup -a AD,ADF,ADR -B -q 40 -Q 18 -C 50 -O u -r LR618874.1 -f tarunm/10_ref/maize.fa --ignore-RG tarunm/20_bam/parent.bam tarunm/20_bam/bulk1.bam tarunm/20_bam/bulk2.bam | bcftools call -vm -f GQ,GP -O u | bcftools filter -i "INFO/MQ>=40" -O z -o tarunm/30_vcf/qtlseq.LR618874.1.vcf.gz >> tarunm/log/bcftools.LR618874.1.log 2>&1

Hello ! First time working with genomic data and this has made my life extremely easy ! just wondering if it would be possible to also add the DP field in the bcftools mpileup so the vcf file can be processed with QTLseqR, or if you have any plans on adding the G statistics that they use papers:

https://acsess.onlinelibrary.wiley.com/doi/10.3835/plantgenome2018.01.0006 (QTLseqR)
https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1002255 (G statistics)

Also not that is needed, since i could probably figure out how to do it, but it would be nice to have an extra python code that would extract the significant regions into a new VCF to be used in snpEff (my genome data is not included, need to crate a new dataset) or if the snpEff option is turned on extract only the significant regions from the txt file that snpEff generates.

hope these comments find you well, and help you improve even more this amazing tool !

Thanks !

MG

I am using version 2.2.2 installed manually and encountering error during vcf generation after successful completion of bam file generation.

the error as follows

File "/home/iipruser/yes/bin/qtlseq", line 33, in
sys.exit(load_entry_point('qtlseq', 'console_scripts', 'qtlseq')())
File "/home/iipruser/QTL-seq/qtlseq/qtlseq.py", line 171, in main
QTLseq(args).run()
File "/home/iipruser/QTL-seq/qtlseq/qtlseq.py", line 166, in run
self.mpileup()
File "/home/iipruser/QTL-seq/qtlseq/qtlseq.py", line 122, in mpileup
mp.run()
File "/home/iipruser/QTL-seq/qtlseq/mpileup.py", line 231, in run
self.concat()
File "/home/iipruser/QTL-seq/qtlseq/mpileup.py", line 182, in concat
sbp.run(cmd1, stdout=sbp.DEVNULL, stderr=sbp.DEVNULL, shell=True, check=True)
File "/home/iipruser/yes/lib/python3.8/subprocess.py", line 512, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command 'cat /media/iipruser/Drive_2/FW_qtlseq_icrisat2/log/bcftools.*.log > /media/iipruser/Drive_2/FW_qtlseq_icrisat2/log/bcftools.log' returned non-zero exit status 2.

pls help me to solve

The code which I have given to run qtlplot with the VCF file: qtlplot -v 30_vcf/qtlseq.vcf -o qtlseq_2.2 -n1 15 -n2 15 -w 1000 -s 500 -S 3 -e Cicer

[QTL-seq:2021-01-18 15:05:23] start to run QTL-plot.
[QTL-seq:2021-01-18 15:05:23] maximum number of threads which you can use is up to 8.
[QTL-seq:2021-01-18 15:05:23] start to run SnpEff.
Traceback (most recent call last):
File "/home/vamshi/miniconda3/bin/qtlplot", line 33, in
sys.exit(load_entry_point('qtlseq', 'console_scripts', 'qtlplot')())
File "/home/vamshi/Downloads/qtlseq/QTL-seq/qtlseq/qtlplot.py", line 207, in main
qp.run()
File "/home/vamshi/Downloads/qtlseq/QTL-seq/qtlseq/qtlplot.py", line 189, in run
self.run_snpEff()
File "/home/vamshi/Downloads/qtlseq/QTL-seq/qtlseq/qtlplot.py", line 46, in run_snpEff
sbp.run(cmd, stdout=sbp.DEVNULL, stderr=sbp.DEVNULL, shell=True, check=True)
File "/home/vamshi/miniconda3/lib/python3.8/subprocess.py", line 512, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command 'snpEff ann -s qtlseq_2.2/snpEff_summary.html SnpEff 30_vcf/qtlseq.vcf 1> qtlseq_2.2/qtlseq.snpEff.vcf 2> qtlseq_2.2/snpEff.log' returned non-zero exit status 255.

It would be great if I had some help solving this issue.
Thanks in advance.

I was working on QTLseq data analysis using YuSugihar/QTL-seq. All process was running smoothly, folder 10_ref, 20_bam, 30_vcf were created. But, suddenly I got an error "PROGRAM KILLED". My queries is inspite of re running the whole program, can I continue it from the same, because is taking much time to make these files.

I have used your package earlier without any issues. But, I am getting this error even if I used the files I had used to successfully generate plots earlier. Please help soon.

[QTL-seq:2020-11-24 03:42:56] start to run QTL-seq.[QTL-seq:2020-11-24 03:42:56] maximum number of threads which you can use is up to 8.
[QTL-seq:2020-11-24 03:42:56] start to index reference fasta.
[QTL-seq:2020-11-24 03:49:38] indexing of reference successfully finished.
[QTL-seq:2020-11-24 03:49:38] start to filter reads.
[QTL-seq:2020-11-24 04:10:35] !!ERROR!! samtools view -b -f 147 -o new_qtlseq_yusugihara_output_F6vsF4/20_bam/bulk2.1000.f147.bam new_qtlseq_yusugihara_output_F6vsF4/20_bam/bulk2.1000.bam >> new_qtlseq_yusugihara_output_F6vsF4/log/samtools.bulk2.1000.f147.log 2>&1
[QTL-seq:2020-11-24 04:10:35] !!ERROR!! samtools view -b -f 83 -o new_qtlseq_yusugihara_output_F6vsF4/20_bam/bulk2.1000.f83.bam new_qtlseq_yusugihara_output_F6vsF4/20_bam/bulk2.1000.bam >> new_qtlseq_yusugihara_output_F6vsF4/log/samtools.bulk2.1000.f83.log 2>&1

I am a Mac M2 chip user. When I use this program on the test files, it goes wrong in the variant calling stage. Could you help me fix the problem? Thanks.
The below is error message:
Process SpawnPoolWorker-3: Traceback (most recent call last): File "/Users/kenhsu/anaconda3/envs/py3.11/lib/python3.11/multiprocessing/process.py", line 314, in _bootstrap self.run() File "/Users/kenhsu/anaconda3/envs/py3.11/lib/python3.11/multiprocessing/process.py", line 108, in run self._target(*self._args, **self._kwargs) File "/Users/kenhsu/anaconda3/envs/py3.11/lib/python3.11/multiprocessing/pool.py", line 114, in worker task = get() ^^^^^ File "/Users/kenhsu/anaconda3/envs/py3.11/lib/python3.11/multiprocessing/queues.py", line 367, in get return _ForkingPickler.loads(res) ^^^^^^^^^^^^^^^^^^^^^^^^^^ File "/Users/kenhsu/QTL-seq/qtlseq/mpileup.py", line 7, in <module> from qtlseq.vcf2index import Vcf2Index File "/Users/kenhsu/QTL-seq/qtlseq/vcf2index.py", line 10, in <module> cache = Manager().dict() ^^^^^^^^^ File "/Users/kenhsu/anaconda3/envs/py3.11/lib/python3.11/multiprocessing/context.py", line 57, in Manager m.start() File "/Users/kenhsu/anaconda3/envs/py3.11/lib/python3.11/multiprocessing/managers.py", line 563, in start self._process.start() File "/Users/kenhsu/anaconda3/envs/py3.11/lib/python3.11/multiprocessing/process.py", line 118, in start assert not _current_process._config.get('daemon'), \ AssertionError: daemonic processes are not allowed to have children

[QTL-seq:2021-06-14 16:33:29] start to run QTL-seq.
[QTL-seq:2021-06-14 16:33:29] maximum number of threads which you can use is up to 16.
[QTL-seq:2021-06-14 16:33:29] start to index reference fasta.
[QTL-seq:2021-06-14 16:38:21] indexing of reference successfully finished.
[QTL-seq:2021-06-14 16:38:21] start to filter reads.
[QTL-seq:2021-06-14 16:58:13] filtering process successfully finished.
[QTL-seq:2021-06-14 16:58:13] start to merge BAMs.
[QTL-seq:2021-06-14 17:14:38] merging process successfully finished.
[QTL-seq:2021-06-14 17:14:38] start to call variants.
[QTL-seq:2021-06-14 17:35:02] variant calling successfully finished.
[QTL-seq:2021-06-14 17:35:02] start to index VCF.
[QTL-seq:2021-06-14 17:35:04] indexing VCF successfully finished.
Traceback (most recent call last):
File "/home/hkplab/anaconda2/bin/qtlplot", line 6, in
from pkg_resources import load_entry_point
File "/home/hkplab/.local/lib/python2.7/site-packages/pkg_resources/init.py", line 3251, in
@_call_aside
File "/home/hkplab/.local/lib/python2.7/site-packages/pkg_resources/init.py", line 3235, in _call_aside
f(*args, **kwargs)
File "/home/hkplab/.local/lib/python2.7/site-packages/pkg_resources/init.py", line 3264, in _initialize_master_working_set
working_set = WorkingSet._build_master()
File "/home/hkplab/.local/lib/python2.7/site-packages/pkg_resources/init.py", line 583, in _build_master
ws.require(requires)
File "/home/hkplab/.local/lib/python2.7/site-packages/pkg_resources/init.py", line 900, in require
needed = self.resolve(parse_requirements(requirements))
File "/home/hkplab/.local/lib/python2.7/site-packages/pkg_resources/init.py", line 786, in resolve
raise DistributionNotFound(req, requirers)
pkg_resources.DistributionNotFound: The 'qtlseq' distribution was not found and is required by the application
[QTL-seq:2021-06-14 17:35:09] QTL-seq successfully finished.

please help me with this error.

QTL-seq package is calling some locations as delta SNP index 1 even if one of the bulks contains the other allele. I have checked the snp.tsv file and found that a much lower depth than available is being used to compute the delta SNP index. The other alleles in this case have base qualities higher than the tool's threshold of MQ> 40 and BQ> 18. Why then are these being left out?