Analysis of ubiome raw data

dada2
(kyeongeui Yun) #1

Good morning,

I have some problem to running the DADA2 plugin.

Sample is pairedend FASTQ file from ubiome company.

Data information,

sample_A_R1.fastq, sample_A_R2.fastq
V4 region, 515F - 806R

I run plugins in qiime2-2018.11.

[Importing]

qiime tools import
–type ‘SampleData[PairedEndSequencesWithQuality]’
–input-path $qiime2_path/manifest.txt
–output-path $import_path/paired-end-demux.qza
–input-format PairedEndFastqManifestPhred33

[CutAdapt]

qiime cutadapt trim-paired
–i-demultiplexed-sequences $import_path/paired-end-demux.qza
–p-cores 4
–p-adapter-f GTGCCAGCMGCCGCGGTAA
–p-front-r GGACTACHVGGGTWTCTAAT
–o-trimmed-sequences $cutadapt_path/paired-end-demux-cutadapt.qza

[DADA2]

qiime dada2 denoise-paired
–i-demultiplexed-seqs $import_path/paired-end-demux-cutadapt.qza
–p-trim-left-f $1
–p-trunc-len-f $2
–p-trim-left-r $3
–p-trunc-len-r $4
–p-n-threads 0
–verbose
–output-dir $qiime2_path/DADA2

:roll_eyes: Error has occured

  1. –p-trim-left-f 10, --p-trim-left-r 10
    –p-trunc-len-f 140, --p-trim-left-140

  2. –p-trim-left-f 10, --p-trim-left-r 10
    –p-tunc-len-f 130, --p-trunc-len-r 130

  3. –p-trim-left-f 10, --p-trim-left-r 10
    –p-trunc-len-f 145, --p-trunc-len-r 145

…
miniconda2/envs/qiime2/lib/python3.5/site-packages/q2_dada2/_denoise.py", line 45, in _check_featureless_table
raise ValueError("No features remain after denoising. Try adjusting "
ValueError: No features remain after denoising. Try adjusting your truncation and trim parameter settings.

Plugin error from dada2:
No features remain after denoising. Try adjusting your truncation and trim parameter settings.

So i change the option in dada2 plugin.

–p-trim-left-f 10, --p-trim-left-r 10
–p-trunc-len-f 150, --p-trunc-len-r 150

…
miniconda2/envs/qiime2/lib/python3.5/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.

Thank you for your time.

(Chris Keefe) #2

Hi @kyeong_yun!
Thanks for writing up a detailed post - unfortunately, your process is not entirely clear to me. A few syntax issues jump out (in 1, multiple trim-lefts, in 1, a --p-*tunc*-left), but I suspect this is not cut/pasted from your actual code.

How did you select your trim and trunc parameters? Can you share the DADA2 stats from the first run (in which the error message suggested you modify the parameters?

qiime metadata tabulate \
  --m-input-file $qiime2_path/DADA2/stats-dada2.qza \
  --o-visualization $qiime2_path/DADA2/stats-dada2.qzv

Best,
Chris

(kyeongeui Yun) #3

Thank you for the prompt reply .

I turned dada2 plugin four times with the above options.

When i turned it like this, the file was not created.

qiime dada2 denoise-paired
–demultiplexed-seqs CutAdapt/paired-end-demux-cutadapt.qza
–p-trimed-left-f 10 --p-trimed-left-r 10
–p-trunc-len-f 140 --p-trunc-len-r 140
–verbose
–o--denoising-stats dada2-stats
–o-table table
–o-representative-sequences rep-seq
–p-n-threads 0

The demultiplexed sequence length has a value between 147 and 150 in paired-end-demux-cutadapt.qzv, the above option are selected.
And quality declined before 7 base.

Thank you so much~ :slight_smile:

(Chris Keefe) #4

@kyeong_yun, sorry if my post was a bit long - if you can share the requested file it will be easier for me to help troubleshoot.