Deblur error with 16S from Novaseq data

[AMosca96 (A.M.)]

Hi everyone,

I am using deblur through QIIME2-2022.8 for 16S data (v3-v4 regions) and I saw many posts about this error message from deblur but, as far I could see, not related to NovaSeq data.:

No sequences passed the filter. It is possible the trim_length (25) may exceed the longest sequence, that all of the sequences are artifacts like PhiX or adapter, or that the positive reference used is not representative of the data being denoised.

Plus, the data quality seems okay, as you can see here:

demux.qzv (306.9 KB)

Here you go my commands (actually, I did many trials setting wider gaps for trimmings):

qiime deblur denoise-16S --i-demultiplexed-seqs demux-filtered.qza --p-trim-length 25 --p-left-trim-len 128 --o-representative-sequences rep-seqs.qza --o-table table.qza --p-sample-stats --o-stats deblur-stats.qza

Could you provide me some info that I'm probably missing?

Thank you so much for your reply in advance!

[colinvwood (Colin Wood)]

Hello @AMosca96 and welcome to the forum,

You are trimming your reads to be no longer than 25 bases long (--p-trim-length 25). This is preventing the forward and reverse reads from being able to merge because they no longer have an overlapping segment.

You are also removing the first 128 bases from the beginning of each read (--p-left-trim-len 128). Taken together, you can see why these parameters are causing an error. You have no bases left in each read.

You probably have the two parameters confused is all.

Thanks.

[AMosca96 (A.M.)]

Hi @colinvwood ,

Thank you for your help! I'm still new in QIIME2 and yeah, the problem is sorted.
But I'm not clear if I should consider the final table with the counts and features as an OTU or ASV table.

[colinvwood (Colin Wood)]

@AMosca96,

They are ASVs.