Dear Qiime developers,
I am running a script to process 7 Miseq runs using the approach explained in the FMT tutorial Fecal microbiota transplant (FMT) study: an exercise — QIIME 2 2019.1.0 documentation.
I first import and check artifical-sequences-free reads using the following :
### Import sequences
# only sequences and properly formatted in the INPUT directory
# in the following directories
RUN1=BP
RUN2=CP
RUN3=DD
RUN4=IB
RUN5=II
RUN6=LU
RUN7=MC
for seqs in ${RUN1} ${RUN2} ${RUN3} ${RUN4} ${RUN5} ${RUN6} ${RUN7}
do
qiime tools import --type SampleData[PairedEndSequencesWithQuality] \
--input-path ${IN}/${seqs} \
--output-path ${IN}/${seqs}_reads.qza \
--input-format CasavaOneEightSingleLanePerSampleDirFmt
Check this artifact to make sure that QIIME now recognizes your data
qiime tools peek ${IN}/${seqs}_reads.qza
### 'Initial' sequence quality control
qiime demux summarize \
--i-data ${IN}/${seqs}_reads.qza \
--o-visualization ${IN}/${seqs}_reads.qzv \
--verbose
done
Then, I run the following loop to run DADA2 independently on the different runs :
### DADA2 workflow
#https://github.com/LangilleLab/microbiome_helper/wiki/Amplicon-SOP-v2-(qiime2-2018.8)
for seqs in ${RUN1} ${RUN2} ${RUN3} ${RUN4} ${RUN5} ${RUN6} ${RUN7}
do
truncF=0
truncL=220
trimF=0
trimL=0
maxee=3
truncq=12
nreadslearn=1000000
chim=consensus
mkdir dada2_output_${seqs}
qiime dada2 denoise-paired --i-demultiplexed-seqs ${IN}/${seqs}_reads.qza \
--p-trunc-len-f ${truncF} \
--p-trunc-len-r ${truncL} \
--p-trim-left-f ${trimF} \
--p-trim-left-r ${trimL} \
--p-max-ee ${maxee} \
--p-trunc-q ${truncq} \
--p-n-reads-learn ${nreadslearn} \
--p-n-threads ${NSLOTS} \
--p-chimera-method ${chim} \
--o-representative-sequences dada2_output_${seqs}/${seqs}_representative_sequences.qza \
--o-table dada2_output_${seqs}/${seqs}_table.qza \
--o-denoising-stats dada2_output_${seqs}/${seqs}_denoising_stats.qza \
--verbose
### Viewing denoising stats
qiime metadata tabulate \
--m-input-file dada2_output_${seqs}/${seqs}_denoising_stats.qza \
--o-visualization dada2_output_${seqs}/${seqs}_denoising_stats.qza
...
It is working like a charm except for the first run and the I got the error :
DADA2 R package version: 1.6.0
- Filtering Error in filterAndTrim(unfiltsF, filtsF, unfiltsR, filtsR, minLen = 175, :
These are the errors (up to 5) encountered in individual cores...
Error in isFALSE(simplify) : could not find function "isFALSE"
Error in isFALSE(simplify) : could not find function "isFALSE"
Error in isFALSE(simplify) : could not find function "isFALSE"
Error in isFALSE(simplify) : could not find function "isFALSE"
Error in isFALSE(simplify) : could not find function "isFALSE"
Execution halted
Traceback (most recent call last):
File "/homedir/constancias/.conda/envs/qiime2-2019.1/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 231, in denoise_paired
run_commands([cmd])
File "/homedir/constancias/.conda/envs/qiime2-2019.1/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
subprocess.run(cmd, check=True)
File "/homedir/constancias/.conda/envs/qiime2-2019.1/lib/python3.6/subprocess.py", line 418, in run
output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command '['run_dada_paired.R', '/tmp/7700394.1.long.q/tmp96myw46k/forward', '/tmp/7700394.1.long.q/tmp96myw46k/reverse', '/tmp/7700394.1.long.q/tmp96myw46k/output.tsv.biom', '/tmp/7700394.1.long.q/tmp96myw46k/track.tsv', '/tmp/7700394.1.long.q/tmp96myw46k/filt_f', '/tmp/7700394.1.long.q/tmp96myw46k/filt_r', '0', '220', '0', '0', '3.0', '12', 'consensus', '1.0', '25', '1000000']' returned non-zero exit status 1.
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/homedir/constancias/.conda/envs/qiime2-2019.1/lib/python3.6/site-packages/q2cli/commands.py", line 274, in call
results = action(**arguments)
File "</homedir/constancias/.conda/envs/qiime2-2019.1/lib/python3.6/site-packages/decorator.py:decorator-gen-442>", line 2, in denoise_paired
File "/homedir/constancias/.conda/envs/qiime2-2019.1/lib/python3.6/site-packages/qiime2/sdk/action.py", line 231, in bound_callable
output_types, provenance)
File "/homedir/constancias/.conda/envs/qiime2-2019.1/lib/python3.6/site-packages/qiime2/sdk/action.py", line 365, in callable_executor
output_views = self._callable(**view_args)
File "/homedir/constancias/.conda/envs/qiime2-2019.1/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 246, in denoise_paired
" and stderr to learn more." % e.returncode)
Exception: An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.
Plugin error from dada2:
An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.
See above for debug info.
Running external command line application(s). This may print messages to stdout and/or stderr.
The command(s) being run are below. These commands cannot be manually re-run as they will depend on temporary files that no longer exist.
Command: run_dada_paired.R /tmp/7700394.1.long.q/tmp96myw46k/forward /tmp/7700394.1.long.q/tmp96myw46k/reverse /tmp/7700394.1.long.q/tmp96myw46k/output.tsv.biom /tmp/7700394.1.long.q/tmp96myw46k/track.tsv /tmp/7700394.1.long.q/tmp96myw46k/filt_f /tmp/7700394.1.long.q/tmp96myw46k/filt_r 0 220 0 0 3.0 12 consensus 1.0 25 1000000
Before running this, I have modified the run_dada_paired.R script in order to include the parameters :
minLen = 175, maxN = 0 in filterAndTrim
MAX_CONSIST=20 in dada(derepRs, selfConsist=TRUE,
Do you have any idea how to solve this issue ?
Thanks a ton