A problom with PEM-Q.py

[zlxlz]

Hello, author！
I ran the example code,but I can't find the FLASH plug-in and I can't read the commands and files that I've given permission to.I don't know how to solve it.
[PEM-Q] genome: mm10
[PEM-Q] sample: CC055c
[PEM-Q] cutsite: 61986726
[PEM-Q] primer: GGAAACCAGAGGGAATCCTC
[PEM-Q] primer_chr: chr15
[PEM-Q] primer_start: 61986633
[PEM-Q] primer_end: 61986652
[PEM-Q] primer_strand: +
mkdir: cannot create directory ‘log’: File exists
######## 01 Reads alignment... ########
align_make_v5.1.py hg38 CC055c_R1.fq.gz CC055c_R2.fq.gz -a CCACGCGTGCTCTACA -p AGGATCTCACCCGGAACAGC -r chr15 -s 61986633 -e 61986652 -d +
[PEM-Q] primerChrom: chr15
[PEM-Q] primer_start: 61986633
[PEM-Q] primer_end: 61986652
[PEM-Q] primer_strand: +
[PEM-Q] primer: AGGATCTCACCCGGAACAGC
[PEM-Q] adapter: CCACGCGTGCTCTACA
[PEM-Q] genome: hg38
[PEM-Q] fastq_r1: CC055c_R1.fq.gz
[PEM-Q] fastq_r2: CC055c_R2.fq.gz
[PEM-Q] adapter alignment file exist, jump...
sh: 1: flash: not found
[PEM-Q] stitching reads using FLASh...
sh: 1: cannot create flash_out/CC055c.merge.fastq: Directory nonexistent
[PEM-Q] merging fastq files...
gzip: flash_out/CC055c.merge.fastq: No such file or directory
mkdir: cannot create directory ‘bwa_align’: File exists
[PEM-Q] index file used /home/zl/bwa/hg38/hg38
[PEM-Q] align to genome...
[PEM-Q] bwa mem -Y -t 8 /home/zl/bwa/hg38/hg38 flash_out/CC055c.merge.fastq.gz > bwa_align/CC055c_sti.sam 2>bwa_align/bwa_alignstich.log
[main_samview] fail to read the header from "bwa_align/CC055c_sti.sam".
[PEM-Q] filter no_primer reads...
[PEM-Q] Your primer sequence: AGGATCTCACCCGGAACAGC
mkdir: cannot create directory ‘primer’: File exists
[PEM-Q] primer position: chr15 1 61986633 61986652
[E::hts_open_format] Failed to open file "bwa_align/CC055c_sti.sort.bam" : No such file or directory
Traceback (most recent call last):
File "/home/zl/PEM-Q/main/align_make_v5.1.py", line 536, in
main()
File "/home/zl/PEM-Q/main/align_make_v5.1.py", line 529, in main
alignment.no_primer_filter()
File "/home/zl/PEM-Q/main/align_make_v5.1.py", line 330, in no_primer_filter
bam_file = pysam.AlignmentFile('bwa_align/'+self.bam_sort, 'rb')
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "pysam/libcalignmentfile.pyx", line 751, in pysam.libcalignmentfile.AlignmentFile.cinit
File "pysam/libcalignmentfile.pyx", line 950, in pysam.libcalignmentfile.AlignmentFile._open
FileNotFoundError: [Errno 2] could not open alignment file bwa_align/CC055c_sti.sort.bam: No such file or directory
######## 02 Barcode Extract... ########
rmb_dedup_v4.py CC055c 17 CCACGCGTGCTCTACA
[PEM-Q] basename: CC055c
[PEM-Q] barcode_length: 17
[PEM-Q] adapter_seqeunce: CCACGCGTGCTCTACA
mkdir: cannot create directory ‘barcode’: File exists
[PEM-Q] align to check adapter...
[PEM-Q] bwa mem -t 8 -k 5 -L 0 -T 14 adapter/adapter flash_out/CC055c.merge.fastq.gz > barcode/CC055c_check.adpt.sam 2>barcode/bwa_align_adapter.log
[PEM-Q] sort and index bam...
[main_samview] fail to read the header from "barcode/CC055c_check.adpt.sam".
[E::hts_open_format] Failed to open file "barcode/CC055c_check.adpt.sort.bam" : No such file or directory
Traceback (most recent call last):
File "/home/zl/PEM-Q/main/rmb_dedup_v4.py", line 327, in
main()
File "/home/zl/PEM-Q/main/rmb_dedup_v4.py", line 322, in main
dedup.filter_multiple_adapter()
File "/home/zl/PEM-Q/main/rmb_dedup_v4.py", line 275, in filter_multiple_adapter
bam_file = pysam.AlignmentFile(self.adapter_bam_check_sort, "rb")#读入sort后的bam文件
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "pysam/libcalignmentfile.pyx", line 751, in pysam.libcalignmentfile.AlignmentFile.cinit
File "pysam/libcalignmentfile.pyx", line 950, in pysam.libcalignmentfile.AlignmentFile._open
FileNotFoundError: [Errno 2] could not open alignment file barcode/CC055c_check.adpt.sort.bam: No such file or directory
######## 03 Define transloc... ########
define_transloc_v5.1_mpf.py CC055c 36573328
[PEM-Q] basename: CC055c
[PEM-Q] cutsite: 36573328
mkdir: cannot create directory ‘transloc’: File exists
mkdir: cannot create directory ‘indel’: File exists
[E::hts_open_format] Failed to open file "barcode/CC055c_sti.filter.sort.bam" : No such file or directory
Traceback (most recent call last):
File "/home/zl/PEM-Q/main/define_transloc_v5.1_mpf.py", line 378, in
main()
File "/home/zl/PEM-Q/main/define_transloc_v5.1_mpf.py", line 372, in main
define_transloc.generate_transloc_tab()
File "/home/zl/PEM-Q/main/define_transloc_v5.1_mpf.py", line 212, in generate_transloc_tab
bam_file = pysam.AlignmentFile(self.bam_sort, 'rb')
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "pysam/libcalignmentfile.pyx", line 751, in pysam.libcalignmentfile.AlignmentFile.cinit
File "pysam/libcalignmentfile.pyx", line 950, in pysam.libcalignmentfile.AlignmentFile._open
FileNotFoundError: [Errno 2] could not open alignment file barcode/CC055c_sti.filter.sort.bam: No such file or directory
######## 04 Define indels... ########
define_indel_v5.1_mpf.py CC055c 36573328 +
[PEM-Q] basename: CC055c
[PEM-Q] cutsite: 36573328
[PEM-Q] primer_strand: +
mkdir: cannot create directory ‘raw’: File exists
[E::hts_open_format] Failed to open file "indel/CC055c_indel.sort.bam" : No such file or directory
Traceback (most recent call last):
File "/home/zl/PEM-Q/main/define_indel_v5.1_mpf.py", line 957, in
main()
File "/home/zl/PEM-Q/main/define_indel_v5.1_mpf.py", line 951, in main
define_indel.generate_indel_tab_file()
File "/home/zl/PEM-Q/main/define_indel_v5.1_mpf.py", line 503, in generate_indel_tab_file
bam_file = pysam.AlignmentFile(self.bam_sort, 'rb')
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "pysam/libcalignmentfile.pyx", line 751, in pysam.libcalignmentfile.AlignmentFile.cinit
File "pysam/libcalignmentfile.pyx", line 950, in pysam.libcalignmentfile.AlignmentFile._open
FileNotFoundError: [Errno 2] could not open alignment file indel/CC055c_indel.sort.bam: No such file or directory
######## 05 DEDUP... ########
dedup_v5.1_mpf.py CC055c
[PEM-Q] basename: CC055c
Traceback (most recent call last):
File "/home/zl/PEM-Q/main/dedup_v5.1_mpf.py", line 156, in
main()
File "/home/zl/PEM-Q/main/dedup_v5.1_mpf.py", line 151, in main
dedup(**kwargs)
File "/home/zl/PEM-Q/main/dedup_v5.1_mpf.py", line 53, in dedup
sid = pd.read_csv("raw/"+basename+"_SID_all.tab",sep = '\t')
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 912, in read_csv
return _read(filepath_or_buffer, kwds)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 577, in _read
parser = TextFileReader(filepath_or_buffer, **kwds)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 1407, in init
self._engine = self._make_engine(f, self.engine)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 1661, in _make_engine
self.handles = get_handle(
^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/common.py", line 859, in get_handle
handle = open(
^^^^^
FileNotFoundError: [Errno 2] No such file or directory: 'raw/CC055c_SID_all.tab'
######## 06 substitutions... ########
define_substitution.py CC055c 36573328 10 +
[PEM-Q] basename: CC055c
[PEM-Q] cutsite: 36573328
[PEM-Q] cutoff: 10
[PEM-Q] primer_strand: +
Traceback (most recent call last):
File "/home/zl/PEM-Q/main/define_substitution.py", line 181, in
main()
File "/home/zl/PEM-Q/main/define_substitution.py", line 176, in main
define_substitution(**kwargs)
File "/home/zl/PEM-Q/main/define_substitution.py", line 66, in define_substitution
data = pd.read_csv(substitution_file, sep = '\t', index_col=False, low_memory=False)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 912, in read_csv
return _read(filepath_or_buffer, kwds)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 577, in _read
parser = TextFileReader(filepath_or_buffer, **kwds)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 1407, in init
self._engine = self._make_engine(f, self.engine)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 1661, in _make_engine
self.handles = get_handle(
^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/common.py", line 859, in get_handle
handle = open(
^^^^^
FileNotFoundError: [Errno 2] No such file or directory: 'unique/CC055c_Substitution.tab'
######## 07 filter and Statistics... ########
define_statistics_add_filter.py CC055c hg38 36573328 500000 AGGATCTCACCCGGAACAGC chr15 + CCACGCGTGCTCTACA
[PEM-Q] basename: CC055c
[PEM-Q] genome: hg38
[PEM-Q] cutsite: 36573328
[PEM-Q] transloc_range: 500000
[PEM-Q] primer: AGGATCTCACCCGGAACAGC
[PEM-Q] primer_chr: chr15
[PEM-Q] primer_strand: +
[PEM-Q] adapter: CCACGCGTGCTCTACA
Processing junction filter and statistics...
mkdir: cannot create directory ‘results’: File exists
Traceback (most recent call last):
File "/home/zl/PEM-Q/main/define_statistics_add_filter.py", line 276, in
main()
File "/home/zl/PEM-Q/main/define_statistics_add_filter.py", line 271, in main
statistics_add_filter(**kwargs)
File "/home/zl/PEM-Q/main/define_statistics_add_filter.py", line 62, in statistics_add_filter
germ = pd.read_csv(germ_file, sep = '\t', index_col=False, low_memory=False)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 912, in read_csv
return _read(filepath_or_buffer, kwds)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 577, in _read
parser = TextFileReader(filepath_or_buffer, **kwds)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 1407, in init
self._engine = self._make_engine(f, self.engine)
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/parsers/readers.py", line 1661, in _make_engine
self.handles = get_handle(
^^^^^^^^^^^
File "/home/zl/anaconda3/lib/python3.11/site-packages/pandas/io/common.py", line 859, in get_handle
handle = open(
^^^^^
FileNotFoundError: [Errno 2] No such file or directory: 'unique/CC055c_Germline_final.tab'
All done, please check log files!
PEM-Q Done in 7.111s

Looking forward to your reply!