'No read passes the filter' in denoising step regardless of truncation parameter

[Wilson]

Hello all,

I am trying to denoise my forward and reverse reads (2x300bp).
My primers are 341f-805r, for 16s V3-V4.
Here are my codes that I've used.

!qiime dada2 denoise-paired
--i-demultiplexed-seqs paired-end-demux.qza
--p-trunc-len-f 260
--p-trunc-len-r 240
--p-trim-left-f 17 \
--p-trim-left-r 21
--o-table table-new.qza
--o-representative-sequences rep-seqs-new.qza
--o-denoising-stats denoising-stats-new.qza

Trimming parameter 17 and 21 was determined based on length of f-primer and r-primer.

However, I got error displaying "No read pass filter". The thing is, the codes above were well-executed 2 days ago, but when I try to re-run today, it displays error.
And now, it displays the error below regardless of a truncation parameter that I choose. It says 'no reads passed the filter' when I choose 0/0, 150/150, 300/300 as truncation parameters, either.

No reads passed the filter. trunc_len_f (260) or trunc_len_r (240) may be individually longer than read lengths, or trunc_len_f + trunc_len_r may be shorter than the length of the amplicon + 12 nucleotides (the length of the overlap).

I additionally attach my quality plot. Plus, what do you think what would be a proper truncation parameter based on the quality plot below? I think forward for 280 and reverse for 220 would be great, but it would be great to hear other's opinion.

[jwdebelius (Justine Debelius)]

Hi @Wilson,

Welcome to the forum!

It's hard to know without getting the stats, unfortunately. I would try running your forward reads alone, and seeing what's getting filtered there. It may not tell you about read joing or chimera slaying, but it will lett you if you're losing reads. (My suspicion is that you're losing reverse reads more than forward, but double check.) I might try cut adapt on your reads instead of primer trimming, you've still got a couple super low quality bases at the begining of your reverse read and that may be why you're not having success.

Best,
Justine

[Wilson]

Thank you for your kind advice