All reads removed after filtering, merging, and quimeras removal with DADA2

[ines]

Hi everyone!

I'm using QIIME2 version 2018.2 to perform bacterial community analysis.

All went well and I got the qzv file after denoising and QC filtering ( file attached). However, when i try to do the filtering, merging and removal of quimeras with DADA2, all reads are removed (sudo docker run -t -i -v $(pwd):/data -w /data Quay qiime dada2 denoise-paired --i-demultiplexed-seqs cutadapt_adapters/trimmed_sequences.qza --p-trunc-len-f 0 --p-trunc-len-r 0 --p-max-ee 2 --p-n-threads 4 --o-representative-sequences seqs-dada2.qza --o-table table-dada2.qza --verbose > stdout-dada2.txt)

I tried changing the parameters - increased number of errors, select trunc at 240 and 200, but it still happens.

Can someone understand what I am doing wrong?

Thank you very very much

sequencesAWARE.qzv (300.1 KB)

[colinbrislawn (Colin J Brislawn)]

Hello Ines,

EDIT: Welcome to the forums!

These look like binned quality scores.

@ines

I'm using QIIME2 version 2018.2

Illumina started using binned quality scores with the Nextseq after this, see DADA2 issue 1307.

I think you are going to need a newer version of Qiime2 to work with this data!
Here's one way to install a newer version: QIIME 2 Library

[ines]

Dear Colin,

Thank you very much for your help.

I installed the newest version of Qiime, however, and even following the pipeline when i get to the filtering and merging point I still loose all the sequences.

I have runned this command: qiime dada2 denoise-paired
--i-demultiplexed-seqs sequencesAWARE-trimmed.qza
--p-trim-left-f 0
--p-trunc-len-f 256
--p-trim-left-r 0
--p-trunc-len-r 256
--o-representative-sequences AWAREasv-seqs.qza
--o-table AWAREasv-table.qza
--o-denoising-stats denoising-stats.qza
--o-base-transition-stats base-transition-stats.qza

[colinvwood (Colin Wood)]

Hello @ines,

Can you post a screenshot of the length distribution tables in the demux visualizer? It might be the case that the your truncation position parameter is causing all reads to be removed.

[ines]

Here it is! Thank you!