Dada2 in Qiime2: losing reads during merging

Dada2 in Qiime2: losing reads during merging

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Hi!

I am trying to use dada2 on my 16S paired-end sequences, from a 2 x 150 bp illumina run. But I am losing almost all my reads at the merging step. The FASTQC shows excellent data quality and no adapters in all my samples.

My pipeline looks like this:

  1. qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path manifest.tsv --output-path pe-demux.qza --input-format PairedEndFastqManifestPhred33V2
  2. qiime dada2 denoise-paired --i-demultiplexed-seqs pe-demux.qza --p-trim-left-f 15 --p-trunc-len-f 130 --p-trunc-len-r 120 --p-n-threads 20 --o-table table.qza --o-representative-sequences rep-seqs.qza --o-denoising-stats denoising-stats.qza &

Fastqc don’t find primers, but I tried p-trim-left parameter in dada2. But still see the same problem of loss of reads during merging.
Finally, all my attempts to run the program give the same result. It is shown in the screenshot. I have only 0-100 merged reads, and only <0.1 of them is non-chimeric. But % of input passed filtered is >95%
enter image description here

I received fast sequencing data and don’t know what adapters were used to prepare the Illumina library.

After unsuccessful attempts with the dada2 program, I tried to use deblur.

  1. qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path manifest.tsv --output-path pe-demux.qza --input-format PairedEndFastqManifestPhred33V2
  2. qiime vsearch join-pairs --i-demultiplexed-seqs ../pe-demux.qza --o-joined-sequences demux-joined.qza
  3. qiime quality-filter q-score --i-demux demux-joined.qza --o-filtered-sequences demux-joined-filtered.qza --o-filter-stats demux-joined-filter-stats.qza
  4. qiime deblur denoise-16S --i-demultiplexed-seqs demux-joined-filtered.qza --p-trim-length 140 --p-sample-stats --p-jobs-to-start 4 --o-representative-sequences rep-seqs.qza --o-table table.qza --o-stats deblur-stats.qza

As a result, I got even worse results than dada2

enter image description here

I would be glad for any advice on merging my readings


Qiime2


Chimer


Dada2

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