Demux graph different sequence lengths

Hi,
I see an error in red under the graph saying "This position (276) is greater than the minimum sequence length observed during subsampling (184 bases). As a result, the plot at this position is not based on data from all of the sequences, so it should be interpreted with caution when compared to plots for other positions". I can understand what it means but how should I intercept it?

I am trying to use the following command for denoising through dada2

qiime dada2 denoise-paired \

--i-demultiplexed-seqs analysis/demux-paired-end.qza
--p-trim-left-f 7
--p-trim-left-r 7
--p-trunc-len-f 298
--p-trunc-len-r 256
--p-n-threads 12
--o-table table.qza
--o-representative-sequences rep-seqs.qza
--o-denoising-stats denoising-stats.qza

Does this parameter seem suitable for the graph? My samples are demultiplexed and trimmed.

Thanks!

demux.qzv (291.6 KB)

This is not an error, it is a warning. Your sequences have different lengths.

dada2 is going to drop any sequences shorter than the truncation length, so you should set the truncation length no longer than the shortest sequence length if you do not want to lose those short reads.

Good luck!

Thanks for clearing that up!

I am new to qiime2 and in the demux summary for forward reads, it’s given that 2% of the reads are of 298nts. Does that mean that 298 is the shortest sequence length (the rest are of 301 nts)?

Thanks!

That is correct

Good luck!

@Shruthi,
Actually, Min len can be shorter than 298, 2% is just second percentile. Looks like the distribution you are looking at does not have min/max length. So I should correct my statement: 2% of your sequences are 298 nt (or less), and the remainder are 301 nt.

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Thanks for clearing that up @Nicholas_Bokulich. So does that mean only an approximate value for the truncation length can be set which is something below 298 nts?

It means that setting a truncation length of 299 nt will cause 2% of your sequences to be dropped.

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@Nicholas_Bokulich Got it thanks!

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