I am trying to trim my Fungal ITS sequences using itsxpress.
The single end sequences are generated by a Pacbio sequel using primer ITS1F and LR3 so most of the sequences length is about 1300bp ,which contain the full ITS region plus partial 18S and 28S region .
I select two sample “S1,S2” to test this tutorial
First I import my sequences:
qiime tools import --input-format SingleEndFastqManifestPhred33V2 --type SampleData[SequencesWithQuality] --input-path manifest.txt --output-path sequences.qza
manifest.txt
sample-id absolute-filepath
S1 $PWD/Sample1.fq.gz
S2 $PWD/Sample2.fq.gz
it totally fine because I check the artifact with
qiime demux summarize
Then I trim the sequences because I want to get a full ITS region sequences without any flanking region:
qiime itsxpress trim-single --i-per-sample-sequences data/sequences.qza --p-region ALL --p-threads 12 --p-cluster-id 1.0 --o-trimmed trimmed.qza
some error happened:
Plugin error from itsxpress:
** Command ‘[‘reformat.sh’, ‘in=/tmp/qiime2-archive-s7ibswhq/0a04cfee-eab7-44cf-a88b-148c9071a7f5/data/S1_0_L001_R1_001.fastq.gz’, ‘out=/tmp/itsxpress_p3vxjmwu/seq_r1.fq.gz’, ‘out2=/tmp/itsxpress_p3vxjmwu/seq_r2.fq.gz’]’ returned non-zero exit status 1.**
Debug info has been saved to /tmp/qiime2-q2cli-err-uzlzrwx9.log
cat /tmp/qiime2-q2cli-err-uzlzrwx9.log
ERROR:root:could not perform read merging with BBmerge. Error from BBmerge was:
java -ea -Xms300m -cp /home/u4040/miniconda3/envs/qiime2-2019.7/opt/bbmap-38.61b-0/current/ jgi.ReformatReads in=/tmp/qiime2-archive-s7ibswhq/0a04cfee-eab7-44cf-a88b-148c9071a7f5/data/S1_0_L001_R1_001.fastq.gz out=/tmp/itsxpress_p3vxjmwu/seq_r1.fq.gz out2=/tmp/itsxpress_p3vxjmwu/seq_r2.fq.gz
Executing jgi.ReformatReads [in=/tmp/qiime2-archive-s7ibswhq/0a04cfee-eab7-44cf-a88b-148c9071a7f5/data/S1_0_L001_R1_001.fastq.gz, out=/tmp/itsxpress_p3vxjmwu/seq_r1.fq.gz, out2=/tmp/itsxpress_p3vxjmwu/seq_r2.fq.gz]
Set INTERLEAVED to true
Input is being processed as paired
Changed from ASCII-33 to ASCII-64 on input ]: 93 -> 62
The ASCII quality encoding offset (64) is not set correctly, or the reads are corrupt; quality value below -5.
Please re-run with the flag ‘qin=33’, ‘ignorebadquality’, or ‘-da’.
Problematic read number 0:
@m54166_190711_083138/4325693/ccs
GCAGTCGAACATGTAGCTGACTCAGGTCACCCGTGTTTCAAGACGGGTCGCTTACGACCATTACGCCAGCGTCCGTGCCGAAGCGCGTTCCTCGGTCCGGGCTGGCCGCATTGCACCCCCGGCTATAAGACGCCCCGAAGGGCGACACATTCCGGGGGCCTTTGACCGACCGCCCAAACCGACGCTGGCCCGCCCGCGGGGGAGTGCACCGGCACGAGTGCCGGCTGAGCCCCCGCGGGCGAGTCTGGTCGCAAGCGCTTCCCTTTCAACAATTTCACGTGCTATTTAACTCTCTTTTCAAAGTGCTTTTCATCTTTCGATCACTCTACTTGTGCGCTATCGGTCTCCGGCCAGTATTTAGCTTTAGATGAAATTTACCACCCATTTAGAGCTGCATTCCCAAACAACTCGACTCGTCGAAGGAGCTTCACACGGACGCGGCCACCCGTCCCAGACGGGATTCTCACCCTCTATGACGGCCCGTTCCAGGGCACTTAGACGGGGGCCGCACCCGAAGCATCCTCTGCAAATTACAACTCGGACCCCGACGGGGCCAGATTTCAAATTTGAGCTCTTGCCGCTTCACTCGCCGTTACTGGGGCAATCCCTGTTGGTTTCTTTTCCTCCGCTTATTGATATGCTTAAGTTCAGCGGGTATCCCTACCTGATCCGAGGTCAACCTGGAAAGAAGTGGTTGACTCGGCGGGCGCCGGCCGGGCCTGCAGAGCGGGTGACAGAGCCCCATACGCTCGAGGACCGGACGCGGCGCCGCCGCTGCCTTTCGGGCCCGTCCCCCCGCGGAGGGCGGGGACGGCGGCCCAACACACAAGCCGTGCTTGAGGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGAGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCGTTGTTGAAAGTTTTCACGATTTCTCACGCGACTCAGACGACACGCCTTCACAGATCAGCGTTCACAGGGTCTCCGGCGGGCACGGGCCGGGGGGACGCCCCCCCGGCGGCCTTGCGGCGGGCCCGCCGAAGCAACACGGTACGGTAGACACGGGTGGGAGGTTGGGCCCCGAAGGACCCGCACTCGGTAATGATCCTTCCGCAGGTTCACCTACGGAAACCTTGTTACGACTTTTACTTCCTCTAAATGACCAAGCTACGATGTGATGCTTGCACAAGTGATCCAGTCGCACGTGTACGAC
+
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@~y~}~~V~~{eK~hj~mw~i~vp{~bt~~~~~~@eRyhyy~{~xzs}}~}~i~~~[~b~q~q~b~~~X~a_~~|[~~~~~~~~~~~}~c~~^~~[a^@qX~~PQ@~jE~o~a~q~y{~~o~~~z~~~~~k~b~aU~~~~@~~~z~~\~~{z~~~~~Y~~~~~~~~~~~~h~~~t~}~~V~~~~~f~~v~~@~~~~~~~~~Y~~~h~~~~~v~~e~~vW~~x~~|w~~~~]~~_~~~~~~J~~~G~~~~i~~~
M~s~p^~vS~z^m~Te_\y~^QzjWv~~J~Z~dg~z~}~j~~~}~~i~tnzw~u~]~Z~wZh~c~g~~~xxAp~xj~UI~~~p~j~kq~~w~x~V~{~iB}yabtO~~~~}~@}~cg~j~Do~t~hZ~n@d~Yx~Gerar}~n~~~~~~~~~~~~~~~~~~@~~~~s~n~md~]~~pS{~t~x~w~_xw~~rTfc}~p~q~v~c~}~U~QoO~p~e~m~ss~]q~`~eubr~rw~H~s~~pL~~}sXm~@~w~sqBmv~T~jZ~jxnPc~D~d~n~us~o~y~{~~~~~{Nz~rxbV~p~hx~~~~~~~~~~~|l^~o~~~~|o~~~~~~~~~m
Offset=64
java.lang.Exception: Aborting.
at shared.KillSwitch.kill(KillSwitch.java:108)
at stream.FASTQ.quadToRead_slow(FASTQ.java:754)
at stream.FASTQ.toReadList(FASTQ.java:625)
at stream.FastqReadInputStream.fillBuffer(FastqReadInputStream.java:107)
at stream.FastqReadInputStream.hasMore(FastqReadInputStream.java:73)
at stream.ConcurrentGenericReadInputStream$ReadThread.readLists(ConcurrentGenericReadInputStream.java:667)
at stream.ConcurrentGenericReadInputStream$ReadThread.run(ConcurrentGenericReadInputStream.java:656)
Traceback (most recent call last):
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/itsxpress/main.py”, line 603, in split_interleaved
p1.check_returncode()
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/subprocess.py”, line 369, in check_returncode
self.stderr)
subprocess.CalledProcessError: Command ‘[‘reformat.sh’, ‘in=/tmp/qiime2-archive-s7ibswhq/0a04cfee-eab7-44cf-a88b-148c9071a7f5/data/S1_0_L001_R1_001.fastq.gz’, ‘out=/tmp/itsxpress_p3vxjmwu/seq_r1.fq.gz’, ‘out2=/tmp/itsxpress_p3vxjmwu/seq_r2.fq.gz’]’ returned non-zero exit status 1.
Traceback (most recent call last):
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/q2cli/commands.py”, line 327, in call
results = action(**arguments)
File “</home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/decorator.py:decorator-gen-277>”, line 2, in trim_single
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 240, in bound_callable
output_types, provenance)
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 383, in callable_executor
output_views = self._callable(**view_args)
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/q2_itsxpress/_itsxpress.py”, line 119, in trim_single
cluster_id=cluster_id)
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/q2_itsxpress/_itsxpress.py”, line 193, in main
threads=threads)
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/q2_itsxpress/_itsxpress.py”, line 72, in _set_fastqs_and_check
tempdir=None)
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/itsxpress/main.py”, line 618, in init
self.split_interleaved()
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/itsxpress/main.py”, line 609, in split_interleaved
raise e
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/itsxpress/main.py”, line 603, in split_interleaved
p1.check_returncode()
File “/home/u4040/miniconda3/envs/qiime2-2019.7/lib/python3.6/subprocess.py”, line 369, in check_returncode
self.stderr)
subprocess.CalledProcessError: Command ‘[‘reformat.sh’, ‘in=/tmp/qiime2-archive-s7ibswhq/0a04cfee-eab7-44cf-a88b-148c9071a7f5/data/S1_0_L001_R1_001.fastq.gz’, ‘out=/tmp/itsxpress_p3vxjmwu/seq_r1.fq.gz’, ‘out2=/tmp/itsxpress_p3vxjmwu/seq_r2.fq.gz’]’ returned non-zero exit status 1.
What should I do bro?