cutadapt trim-paired

amin · April 7, 2022, 2:45pm

Hi,
I am having problem while running the code:
qiime cutadapt trim-paired --i-demultiplexed-sequences demux.qza --p-cores 16 --p-adapter-f AAGCTCGTAGTTGAATTTCG --p-adapter-r CCCAACTATCCCTATTAATCAT --o-trimmed-sequences primer-trimmed.qza --verbose

I use qiime2-2021.8 and the primer is from 18S rRNA gene. The part of error I am getting is :

Sequence: CCCAACTATCCCTATTAATCAT; Type: regular 3'; Length: 22; Trimmed: 1 times

No. of allowed errors:
1-9 bp: 0; 10-19 bp: 1; 20-22 bp: 2

Bases preceding removed adapters:
A: 0.0%
C: 0.0%
G: 0.0%
T: 0.0%
none/other: 100.0%

Overview of removed sequences
length count expect max.err error counts
258 1 0.0 2 1

Command: cutadapt --cores 16 --error-rate 0.1 --times 1 --overlap 3 --minimum-length 1 -o /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-d_vc7i7h/CK3-1-AMV45F-AMDGR_20_L001_R1_001.fastq.gz -p /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-d_vc7i7h/CK3-1-AMV45F-AMDGR_21_L001_R2_001.fastq.gz --adapter AAGCTCGTAGTTGAATTTCG -A CCCAACTATCCCTATTAATCAT /tmp/qiime2-archive-wlq8h42a/cde7219d-8e9d-4ab9-9c4b-f5971ee7f12a/data/CK3-1-AMV45F-AMDGR_20_L001_R1_001.fastq.gz /tmp/qiime2-archive-wlq8h42a/cde7219d-8e9d-4ab9-9c4b-f5971ee7f12a/data/CK3-1-AMV45F-AMDGR_21_L001_R2_001.fastq.gz

This is cutadapt 3.4 with Python 3.8.10
Command line parameters: --cores 16 --error-rate 0.1 --times 1 --overlap 3 --minimum-length 1 -o /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-d_vc7i7h/CK3-1-AMV45F-AMDGR_20_L001_R1_001.fastq.gz -p /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-d_vc7i7h/CK3-1-AMV45F-AMDGR_21_L001_R2_001.fastq.gz --adapter AAGCTCGTAGTTGAATTTCG -A CCCAACTATCCCTATTAATCAT /tmp/qiime2-archive-wlq8h42a/cde7219d-8e9d-4ab9-9c4b-f5971ee7f12a/data/CK3-1-AMV45F-AMDGR_20_L001_R1_001.fastq.gz /tmp/qiime2-archive-wlq8h42a/cde7219d-8e9d-4ab9-9c4b-f5971ee7f12a/data/CK3-1-AMV45F-AMDGR_21_L001_R2_001.fastq.gz
Processing reads on 16 cores in paired-end mode ...
ERROR: Traceback (most recent call last):
File "/home/aferdous/conda/envs/qiime2-2021.8/lib/python3.8/site-packages/cutadapt/pipeline.py", line 631, in run
(n, bp1, bp2) = self._pipeline.process_reads()
File "/home/aferdous/conda/envs/qiime2-2021.8/lib/python3.8/site-packages/cutadapt/pipeline.py", line 428, in process_reads
for read1, read2 in self._reader:
File "/home/aferdous/conda/envs/qiime2-2021.8/lib/python3.8/site-packages/dnaio/init.py", line 285, in iter
raise FileFormatError(
dnaio.exceptions.FileFormatError: Error in sequence file at unknown line: Reads are improperly paired. Read name 'M02696:121:000000000-JJMWP:1:1101:12051:1102 1:N:0:173' in file 1 does not match 'M02696:121:000000000-JJMWP:1:1101:20412:1129 2:N:0:174' in file 2.

ERROR: Traceback (most recent call last):
File "/home/aferdous/conda/envs/qiime2-2021.8/lib/python3.8/site-packages/cutadapt/pipeline.py", line 555, in run
for chunk_index, (chunk1, chunk2) in enumerate(
File "/home/aferdous/conda/envs/qiime2-2021.8/lib/python3.8/site-packages/dnaio/chunks.py", line 117, in read_paired_chunks
raise ValueError("FASTQ record does not fit into buffer")
ValueError: FASTQ record does not fit into buffer

cutadapt: error: Error in sequence file at unknown line: Reads are improperly paired. Read name 'M02696:121:000000000-JJMWP:1:1101:12051:1102 1:N:0:173' in file 1 does not match 'M02696:121:000000000-JJMWP:1:1101:20412:1129 2:N:0:174' in file 2.
Traceback (most recent call last):
File "/home/aferdous/conda/envs/qiime2-2021.8/lib/python3.8/site-packages/q2cli/commands.py", line 329, in call
results = action(**arguments)
File "", line 2, in trim_paired
File "/home/aferdous/conda/envs/qiime2-2021.8/lib/python3.8/site-packages/qiime2/sdk/action.py", line 245, in bound_callable
outputs = self.callable_executor(scope, callable_args,
File "/home/aferdous/conda/envs/qiime2-2021.8/lib/python3.8/site-packages/qiime2/sdk/action.py", line 391, in callable_executor
output_views = self._callable(**view_args)
File "/home/aferdous/conda/envs/qiime2-2021.8/lib/python3.8/site-packages/q2_cutadapt/_trim.py", line 189, in trim_paired
run_commands(cmds)
File "/home/aferdous/conda/envs/qiime2-2021.8/lib/python3.8/site-packages/q2_cutadapt/_trim.py", line 30, in run_commands
subprocess.run(cmd, check=True)
File "/home/aferdous/conda/envs/qiime2-2021.8/lib/python3.8/subprocess.py", line 516, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['cutadapt', '--cores', '16', '--error-rate', '0.1', '--times', '1', '--overlap', '3', '--minimum-length', '1', '-o', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-d_vc7i7h/CK3-1-AMV45F-AMDGR_20_L001_R1_001.fastq.gz', '-p', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-d_vc7i7h/CK3-1-AMV45F-AMDGR_21_L001_R2_001.fastq.gz', '--adapter', 'AAGCTCGTAGTTGAATTTCG', '-A', 'CCCAACTATCCCTATTAATCAT', '/tmp/qiime2-archive-wlq8h42a/cde7219d-8e9d-4ab9-9c4b-f5971ee7f12a/data/CK3-1-AMV45F-AMDGR_20_L001_R1_001.fastq.gz', '/tmp/qiime2-archive-wlq8h42a/cde7219d-8e9d-4ab9-9c4b-f5971ee7f12a/data/CK3-1-AMV45F-AMDGR_21_L001_R2_001.fastq.gz']' returned non-zero exit status 1.

Plugin error from cutadapt:

Command '['cutadapt', '--cores', '16', '--error-rate', '0.1', '--times', '1', '--overlap', '3', '--minimum-length', '1', '-o', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-d_vc7i7h/CK3-1-AMV45F-AMDGR_20_L001_R1_001.fastq.gz', '-p', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-d_vc7i7h/CK3-1-AMV45F-AMDGR_21_L001_R2_001.fastq.gz', '--adapter', 'AAGCTCGTAGTTGAATTTCG', '-A', 'CCCAACTATCCCTATTAATCAT', '/tmp/qiime2-archive-wlq8h42a/cde7219d-8e9d-4ab9-9c4b-f5971ee7f12a/data/CK3-1-AMV45F-AMDGR_20_L001_R1_001.fastq.gz', '/tmp/qiime2-archive-wlq8h42a/cde7219d-8e9d-4ab9-9c4b-f5971ee7f12a/data/CK3-1-AMV45F-AMDGR_21_L001_R2_001.fastq.gz']' returned non-zero exit status 1.

Since I received an analysis on this data from sequencing facility using their own pipeline (not qiime2), I assume there is no error in sequencing. But I tried all the possible suggestions from previous problems in this forum. Eagerly waiting for your kind response.

Regards,
Amin

colinbrislawn · April 8, 2022, 12:38am

Hello @amin,

Here's the core of the error:

But... it looks like there is an error in pairing, at least from the perspective of q2-cutadapt.

Could your sequencing facility send you the raw data? If not, we have other options, but that's the best and easiest one so let's start there!

amin · April 8, 2022, 2:24am

Hi @colinbrislawn ,
You are right, I just realized I made a mistake in my manifest.csv file while making demux.qza file that might cause error in pairing. I will update you once I fix it. I really appreciate your prompt response; this is the amazing part of qiime2forum, every time I am stuck, you save me.

Regards,
Amin

amin · April 10, 2022, 11:09pm

Solved, Thank you so much.

system · May 12, 2022, 5:10am

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