low proportion of merged sequences using the DADA2

Technical Support
1

Hi all.

I am testing my 16S data using DADA2 on 2x250 paired end reads. This was the quality score box plots.
qual_viz.qzv (309.8 KB)

Based on the results, the sequences were denoised with DADA2.

The script i used:

qiime dada2 denoise-paired --i-demultiplexed-seqs 16s_cb_paired-end-demux.qza --o-table table --o-representative-sequences representative_sequences --p-trunc-len-f 240 --p-trunc-len-r 240 --p-trim-left-f 20 --p-trim-left-r 20 --o-denoising-stats denoising-stats.qza

This was the denoising stats
stats.tsv (3.5 KB)

It looks like most of our sequences were dropped out because they fail to merge.

I wonder why so many reads were lost especially for sample A7, M1, and F1.

Thanks in advance.

2

Hello!
Indeed, you are loosing tons reads at merging step. Since there are no issues with filtering step, most likely overlapping region is too short. I would suggest to:

  • set truncation parameter to 0, which will disable it
  • add option to decrease min overlap (check docs how to do it).

Best,

3

Hi, timanix!
Really appreciate for your reply! We will try the methods you suggested.

Best wishes

4

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