DADA2 command failing in Qiime 2022.2

Hi,

My DADA2 pipeline of Qiime2 pipeline fails repeatedly.

Qiime 2 instllation- Virtual MAchine
Version- Qiime 2 2022.2
command I used is - qiime dada2 denoise-paired
--i-demultiplexed-seqs pe_reads_cutadapt_trimmed.qza
--p-trim-left-f 50
--p-trim-left-r 13
--p-trunc-len-f 212
--p-trunc-len-r 194
--output-dir DADA2_denoising_output
--verbose
&> DADA2_denoising.log

The quality drops below 30 hence choose these values

pe_reads_cutadapt_trimmed.qzv (330.6 KB)

Error log-R version 4.1.3 (2022-03-10)
Loading required package: Rcpp
DADA2: 1.22.0 / Rcpp: 1.0.8.3 / RcppParallel: 5.1.5

  1. Filtering The filter removed all reads: /tmp/tmp2j6gy25b/filt_f/2-B12-BAL19_S120_L001_R1_001.fastq.gz and /tmp/tmp2j6gy25b/filt_r/2-B12-BAL19_S120_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmp2j6gy25b/filt_f/2-C03-BAL22_S123_L001_R1_001.fastq.gz and /tmp/tmp2j6gy25b/filt_r/2-C03-BAL22_S123_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmp2j6gy25b/filt_f/2-C09-BAL28_S129_L001_R1_001.fastq.gz and /tmp/tmp2j6gy25b/filt_r/2-C09-BAL28_S129_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmp2j6gy25b/filt_f/2-C11-BAL30_S131_L001_R1_001.fastq.gz and /tmp/tmp2j6gy25b/filt_r/2-C11-BAL30_S131_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmp2j6gy25b/filt_f/2-D02-BAL33_S134_L001_R1_001.fastq.gz and /tmp/tmp2j6gy25b/filt_r/2-D02-BAL33_S134_L001_R2_001.fastq.gz not written.
    Some input samples had no reads pass the filter.
    .......................................................................................................................x..x....x.x..x.
  2. Learning Error Rates
    167395572 total bases in 1033306 reads from 12 samples will be used for learning the error rates.

Uploading: pe_reads_cutadapt_trimmed.qza...

Here are all the codes I ran

data importing

qiime tools import
--type 'SampleData[PairedEndSequencesWithQuality]'
--input-path /media/sf_Jessica/fqgz
--input-format CasavaOneEightSingleLanePerSampleDirFmt
--output-path paired-end-demux.qza

2.2 primer trimming with cutadapt
ref GitHub - SchlossLab/MiSeq_WetLab_SOP

DNA was amplified by using the 515f/806r primer set:
Forward V4: GTGCCAGCMGCCGCGGTAA
Reverse V4: GGACTACHVGGGTWTCTAAT

qiime cutadapt trim-paired
--i-demultiplexed-sequences paired-end-demux.qza
--p-cores 20
--p-front-f GTGCCAGCMGCCGCGGTAA
--p-front-r GGACTACHVGGGTWTCTAAT
--o-trimmed-sequences pe_reads_cutadapt_trimmed.qza
--verbose
&> primer_trim.log

2.3 Summarize trimmed FASTQs
`
qiime demux summarize
--i-data pe_reads_cutadapt_trimmed.qza
--o-visualization pe_reads_cutadapt_trimmed.qzv

2.4 DADA2 denoising
2.4.1 DADA2 pipeline
code in qiime2:

qiime dada2 denoise-paired
--i-demultiplexed-seqs pe_reads_cutadapt_trimmed.qza
--p-trim-left-f 50
--p-trim-left-r 13
--p-trunc-len-f 212
--p-trunc-len-r 194
--output-dir DADA2_denoising_output
--verbose
&> DADA2_denoising.log

Sorry for the multiple entries. I apologize. Here is the metadata
N2_mappingfile_2023.csv (25.0 KB)

Hello @Akhilav1,

I would remove the samples are that aren't passing the filter and try again. If you look at the demux visualizer you can see that the ones failing have a very low read count.

2 Likes

Hi,

Thank you for the advise. I have a couple of question. The samples that did not pass the filter have forward and reverse sequence score
BAL 19 -36
BAL 22 - 3
BAL 28 - 102
BAL 30 - 6
BAL 33 - 4

  1. Should I target to remove all reads less than counts 150?
  2. Almost 21 samples have read count less that 10000. Should I remove all of them?
  3. Is there a code to remove the sequences from the study or should I remove them manually? I believe the sequences that didn't meet minumum sequence count will be dropped automatically.

Please let me know. Appreciate your help.

Thanks,
Akhil

Hello @Akhilav1,

Should I target to remove all reads less than counts 150?
Almost 21 samples have read count less that 10000. Should I remove all of them?

It looks like there is a big jump in coverage between the 2-C02-BAL21 sample with 7k reads and the 2-A07-BAL2 sample with only 300 reads. I would probably remove everything below this step down.

  1. Is there a code to remove the sequences from the study or should I remove them manually? I believe the sequences that didn't meet minumum sequence count will be dropped automatically.

Yes, you can use qiime demux filter-samples.

2 Likes

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