Using READemption v2.0.3
Besides the very long time it takes to build fragments, I have warnings because of the missing index files for bam files. Then errors occur due to "Too many open files".
total mapped (%) unique (%) multi (%) split (%)
all 45714906 44783193 97.96% 4687693 10.25% 40095500 87.71% 0 0.00%
pair 22857453 21504215 94.08% 2033415 8.90% 19470800 85.18% 0 0.00%
[SEGEMEHL] Tue Jul 11 12:34:39 2023:
Goodbye.
"Die Ficker!" (Thommy)
controller align_paired_end_reads stop 2023-07-11 13:51:57.318376
controller generate_read_alignment_stats start 2023-07-11 13:51:57.318614
readalignerstats count_aligned_reads_and_alignments start 2023-07-11 13:51:57.764202
readalignerstats count_aligned_reads_and_alignments start 2023-07-11 13:51:57.764462
readalignerstats count_aligned_reads_and_alignments start 2023-07-11 13:51:57.764490
readalignerstats count_aligned_reads_and_alignments start 2023-07-11 13:51:57.768954
readalignerstats count_aligned_reads_and_alignments stop 2023-07-12 13:42:39.552076
readalignerstats count_unaligned_reads start 2023-07-12 13:42:39.610802
readalignerstats count_unaligned_reads stop 2023-07-12 13:42:40.274719
readalignerstats count_aligned_reads_and_alignments stop 2023-07-12 16:58:52.556453
readalignerstats count_unaligned_reads start 2023-07-12 16:58:52.559282
readalignerstats count_unaligned_reads stop 2023-07-12 16:58:54.523031
readalignerstats count_aligned_reads_and_alignments stop 2023-07-12 18:13:50.040636
readalignerstats count_unaligned_reads start 2023-07-12 18:13:50.042781
readalignerstats count_unaligned_reads stop 2023-07-12 18:13:51.502212
readalignerstats count_aligned_reads_and_alignments stop 2023-07-12 18:39:25.329928
readalignerstats count_unaligned_reads start 2023-07-12 18:39:25.332696
readalignerstats count_unaligned_reads stop 2023-07-12 18:39:26.450343
controller generate_read_alignment_stats stop 2023-07-12 18:39:27.672006
controller sort bams by name and index start 2023-07-12 19:35:14.979915
controller sort bams by name and index end 2023-07-12 21:12:23.925582
controller build_fragments start 2023-07-12 21:12:23.925959
[E::idx_find_and_load] [E::idx_find_and_load] [E::idx_find_and_load] Could not retrieve index file for 'READemption_analysis_NEBNext-Ultra-II_kit/
output/align/alignments/NEBNext-Ultra-II_kit_RNA-Seq_OD_0.2_rep1_alignments_final_sorted.bam'Could not retrieve index file for 'READemption_analys
is_NEBNext-Ultra-II_kit/output/align/alignments/NEBNext-Ultra-II_kit_RNA-Seq_OD_2.0_rep2_alignments_final_sorted.bam'Could not retrieve index file
for 'READemption_analysis_NEBNext-Ultra-II_kit/output/align/alignments/NEBNext-Ultra-II_kit_RNA-Seq_OD_2.0_rep1_alignments_final_sorted.bam'
[E::idx_find_and_load] Could not retrieve index file for 'READemption_analysis_NEBNext-Ultra-II_kit/output/align/alignments/NEBNext-Ultra-II_kit_R
NA-Seq_OD_0.2_rep2_alignments_final_sorted.bam'
[E::hts_open_format] Failed to open file "READemption_analysis_NEBNext-Ultra-II_kit/output/align/alignments/NEBNext-Ultra-II_kit_RNA-Seq_OD_0.2_re
p1_alignments_final_fragments.bam_sorted.tmp.1009.bam" : Too many open files
[E::hts_open_format] Failed to open file "READemption_analysis_NEBNext-Ultra-II_kit/output/align/alignments/NEBNext-Ultra-II_kit_RNA-Seq_OD_2.0_re
p2_alignments_final_fragments.bam_sorted.tmp.1005.bam" : Too many open files
[E::hts_open_format] Failed to open file "READemption_analysis_NEBNext-Ultra-II_kit/output/align/alignments/NEBNext-Ultra-II_kit_RNA-Seq_OD_0.2_re
p2_alignments_final_fragments.bam_sorted.tmp.1007.bam" : Too many open files
[E::hts_open_format] Failed to open file "READemption_analysis_NEBNext-Ultra-II_kit/output/align/alignments/NEBNext-Ultra-II_kit_RNA-Seq_OD_2.0_re
p1_alignments_final_fragments.bam_sorted.tmp.1003.bam" : Too many open files
concurrent.futures.process._RemoteTraceback:
"""
Traceback (most recent call last):
File "/usr/lib/python3.10/concurrent/futures/process.py", line 246, in _process_worker
r = call_item.fn(*call_item.args, **call_item.kwargs)
File "/usr/local/lib/python3.10/dist-packages/reademptionlib/fragmentbuilder.py", line 113, in build_bam_file_with_fragments
pysam.sort("-o", tmp_sorted_outfile, fragment_alignment_path)
File "/home/muhammad/.local/lib/python3.10/site-packages/pysam/utils.py", line 69, in __call__
raise SamtoolsError(
pysam.utils.SamtoolsError: 'samtools returned with error 1: stdout=, stderr=[bam_sort_core] merging from 6420 files and 1 in-memory blocks...\nsam
tools sort: fail to open "READemption_analysis_NEBNext-Ultra-II_kit/output/align/alignments/NEBNext-Ultra-II_kit_RNA-Seq_OD_0.2_rep2_alignments_fi
nal_fragments.bam_sorted.tmp.1007.bam": Too many open files\n'
"""
The above exception was the direct cause of the following exception:
Traceback (most recent call last):
File "/usr/local/bin/reademption", line 715, in <module>
main()
File "/usr/local/bin/reademption", line 22, in main
args.func(controller)
File "/usr/local/bin/reademption", line 687, in align_reads
controller.align_reads()
File "/usr/local/lib/python3.10/dist-packages/reademptionlib/controller.py", line 212, in align_reads
self._build_fragments()
File "/usr/local/lib/python3.10/dist-packages/reademptionlib/controller.py", line 298, in _build_fragments
self._check_job_completeness(jobs)
File "/usr/local/lib/python3.10/dist-packages/reademptionlib/controller.py", line 801, in _check_job_completeness
raise (job.exception())
pysam.utils.SamtoolsError: 'samtools returned with error 1: stdout=, stderr=[bam_sort_core] merging from 6420 files and 1 in-memory blocks...\nsam
tools sort: fail to open "READemption_analysis_NEBNext-Ultra-II_kit/output/align/alignments/NEBNext-Ultra-II_kit_RNA-Seq_OD_0.2_rep2_alignments_fi
nal_fragments.bam_sorted.tmp.1007.bam": Too many open files\n'
Using READemption v2.0.3 Besides the very long time it takes to build fragments, I have warnings because of the missing index files for bam files. Then errors occur due to "Too many open files".