I am using q2-itsxpress to handle my full-length fungi Pacbio CCS.
The bug showed in BBmap merge step:
qiime itsxpress trim-single --i-per-sample-sequences rawdata/demux.qza --p-region ALL --o-trimmed test.qza --verbose
ERROR:root:could not perform read merging with BBmerge. Error from BBmerge was:
java -ea -Xms300m -cp /home/test/miniconda3/envs/qiime2-2020.8/opt/bbmap-38.69-0/current/ jgi.ReformatReads in=/tmp/qiime2-archive-dqnjht7w/d5b51a90-b232-4a1f-a88d-4a37e382e0e3/data/DJ0h1.fastq.gz_10_L001_R1_001.fastq.gz out=/tmp/itsxpress_d0tb7kep/seq_r1.fq.gz out2=/tmp/itsxpress_d0tb7kep/seq_r2.fq.gz
Executing jgi.ReformatReads [in=/tmp/qiime2-archive-dqnjht7w/d5b51a90-b232-4a1f-a88d-4a37e382e0e3/data/DJ0h1.fastq.gz_10_L001_R1_001.fastq.gz, out=/tmp/itsxpress_d0tb7kep/seq_r1.fq.gz, out2=/tmp/itsxpress_d0tb7kep/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_083138/4260081/ccs
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACTGTGATTTACTACTACACTGCGTGAGCGGAACGAAAACAACAACACCTAAAATGTGGAATATAGCATATAGTCGACAAGAGAAATCTACGAAAAACAAACAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAGCGCAGCGAAATGCGATACCTAGTGTGAATTGCAGCCATCGTGAATCATCGAGTTCTTGAACGCACATTGCGCCCCTCGGCATTCCGGGGGGCATGCCTGTTTGAGCGTCGTTTCCATCTTGCGCGTGCGCAGAGTTGGGGGAGCGGAGCGGACGACGTGTAAAGAGCGTCGGAGCTGCGACTCGCCTGAAAGGGAGCGAAGCTGGCCGAGCGAACTAGACTTTTTTTCAGGGACGCTTGGCGGCCGAGAGCGAGTGTTGCGAGACAACAAAAAGCTCGACCTCAAATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCCTCAGTAGCGGCGAGTGAAGCGGCAAGAGCTCAGATTTGAAATCGTGCTTTGCGGCACGAGTTGTAGATTGCAGGTTGGAGTCTGTGTGGAAGGCGGTGTCCAAGTCCCTTGGAACAGGGCGCCCAGGAGGGTGAGAGCCCCGTGGGATGCCGGCGGAAGCAGTGAGGCCCTTCTGACGAGTCGAGTTGTTTGGGAATGCAGCTCCAAGCGGGTGGTAAATTCCATCTAAGGCTAAATACTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGCACGTGAAATTGTTGAAAGGGAAGGGTATGCGATTAGCGGCCAGCAGGAGGTGCCTTCTCGTGAAAAGGCCGTGCACCGTCTTCGGACACCGTGCGCGGAGATGGCGAGGGGGCGCCTGAGGTCTGCGAACTCGAGGTTGCTGGCGTAATGATTGCATACCA
+
@@@@@@@@@@@@@@@@@}bstq~_iH~@~IYVs~P~P~Hqtm~aconp]
t|qGin\mjmjXdYj^Ion\KMbsN~Ro^~AmxHg{FnglJR}l}@Rrb~@]PE_Taminsq]qqvjrda|U[[m|~@stnst]~r@Z~b@f]nv@k{x@q~Sm~IS~OB_b^q~a~axOZlouujtX\tqs~AkslXessrutt~JquuutXs~qqqfsm~b~LSmookGjitsalx@ttpZo\qm~PsiCT~Kdstqrq~Xgar~~~MZs~NCT~b~Y~~~~~@sUdm~b[h~~@jsruZHYn~~CtR
lmHnuusjstuuuqrrm|Kbvwe@rqu~YHgu~RU]VthX@^pr~Ea@hXXlZ|ZVhI]tuusrsuqx@cZ~~T~eIZndWuVprr~a~busqdu~RmqqqLm|~~~~~@kUx~@]ktq~_~ai~Z~aEkirquusphs~Xtptsqth~@hiu~~ChmJg]t~XWog~@tvn~Jmqn_rZt^~~Wlnk^~V
~c@ramfgvf~[
~su~
u~~~~@s~~JcF~atp~~Wu~
u~Css]qsng[vKLebirv~aov~Np{S]pf[budsXUv@]biakx~@p[~NWTkt^p~\Iiuqsraaun~a~~
tnt[ntqtm~O~akWs~@leo~a~qu~tG~@s~Hlr~~Htu~~Fk~Mr~~Wnpr]u]d~~@mq~~Qtqr~\~^vt@~Vquqpttq~@~wK~Vqquvs^f[dVQgVn@Ump@V
LnHPHktrs~b~
qs~{HkwZZ~~J~be@stq^~~rq~~Vssam~Gt]W]eorq\tctobtu~Rl~OppjXlqruup~b~~Tququ~~~JqrkU~~Tu~~~Aqnnhrtt~~@lqrurr]ltjoC|FT@@@[q@cj@a@j~OYtUguZ[r@otu~a~Tkq]j~Isc@MPu_zYjdtalii|~A~_~QrbGs^~
uuq~b^d@ee^zTIgstnr~MsZts~urZx~~~Duu~
oq[pU\qov[ts]YgrjpxYpGtnLY@u\s~Nquu~duusrIg[q
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/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/itsxpress/main.py", line 586, in split_interleaved
p1.check_returncode()
File "/home/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/subprocess.py", line 389, in check_returncode
self.stderr)
subprocess.CalledProcessError: Command '['reformat.sh', 'in=/tmp/qiime2-archive-dqnjht7w/d5b51a90-b232-4a1f-a88d-4a37e382e0e3/data/DJ0h1.fastq.gz_10_L001_R1_001.fastq.gz', 'out=/tmp/itsxpress_d0tb7kep/seq_r1.fq.gz', 'out2=/tmp/itsxpress_d0tb7kep/seq_r2.fq.gz']' returned non-zero exit status 1.
Traceback (most recent call last):
File "/home/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/q2cli/commands.py", line 329, in call
results = action(**arguments)
File "", line 2, in trim_single
File "/home/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/qiime2/sdk/action.py", line 245, in bound_callable
output_types, provenance)
File "/home/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/qiime2/sdk/action.py", line 390, in callable_executor
output_views = self._callable(**view_args)
File "/home/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/q2_itsxpress/_itsxpress.py", line 123, in trim_single
cluster_id=cluster_id)
File "/home/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/q2_itsxpress/_itsxpress.py", line 203, in main
threads=threads)
File "/home/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/q2_itsxpress/_itsxpress.py", line 74, in _set_fastqs_and_check
reversed_primers=reversed_primers)
File "/home/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/itsxpress/main.py", line 603, in init
self.split_interleaved(reversed_primers=reversed_primers)
File "/home/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/itsxpress/main.py", line 596, in split_interleaved
raise e
File "/home/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/itsxpress/main.py", line 586, in split_interleaved
p1.check_returncode()
File "/home/test/miniconda3/envs/qiime2-2020.8/lib/python3.6/subprocess.py", line 389, in check_returncode
self.stderr)
subprocess.CalledProcessError: Command '['reformat.sh', 'in=/tmp/qiime2-archive-dqnjht7w/d5b51a90-b232-4a1f-a88d-4a37e382e0e3/data/DJ0h1.fastq.gz_10_L001_R1_001.fastq.gz', 'out=/tmp/itsxpress_d0tb7kep/seq_r1.fq.gz', 'out2=/tmp/itsxpress_d0tb7kep/seq_r2.fq.gz']' returned non-zero exit status 1.
Plugin error from itsxpress:
Command '['reformat.sh', 'in=/tmp/qiime2-archive-dqnjht7w/d5b51a90-b232-4a1f-a88d-4a37e382e0e3/data/DJ0h1.fastq.gz_10_L001_R1_001.fastq.gz', 'out=/tmp/itsxpress_d0tb7kep/seq_r1.fq.gz', 'out2=/tmp/itsxpress_d0tb7kep/seq_r2.fq.gz']' returned non-zero exit status 1.
See above for debug info.
I guess bbtools can not handle Pacbio Phred-33 right now