Few sequences were left after filtering

Dear All,

Can anyone advise me about this issue? After running the command below

qiime dada2 denoise-paired
–i-demultiplexed-seqs trimmed_seqs_bacteria.qza
–p-trim-left-f 0
–p-trim-left-r 0
–p-trunc-len-f 228
–p-trunc-len-r 206
–o-table table_bacteria_dada206.qza
–o-representative-sequences rep_seqs_bacteria_dada206.qza
–o-denoising-stats denoising_stats_bacteria_dada206.qza

I got only few merged sequences. Would it be possible to adjust the command to get more sequences? Would it have any influence on the results if I don't adjust the command?The amplicon region is V4V5, and I ran 2x250bp on miseq platform.


denoising_stats_bacteria_dada206.qzv (1.2 MB) table_bacteria_dada206.qzv (667.2 KB) trimmed_seqs_bacteria.qzv (296.3 KB)

Hi @hesongbing!
These are very frequently asked questions here on the forum, and understanding why this problem is happening will be useful and important for future analyses. I’d encourage you to do some reading :mag: about DADA2 trim and trunc parameters, and why people lose sequences during denoising.

These questions may help guide you:

Do you know how to interpret your denoising stats table? That tool can tell you why you are losing so many sequences.

Do you know how to calculate the minimum number of b.p. required for DADA2 to join reads?
(Hint: In order to do this, you’ll need to know how long your target V4-V5 amplicon is).

Let me know if you have any specific questions about your reading - I’m happy to help.

Chris :snail:

As an aside, the quality scores plotted in your trimmed_seqs_bacteria.qzv look weirdly clean. Usually there’s more noise in those plots - are you (or your sequencing center) preprocessing the reads in a way that impacts quality scores?

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