quality score and where to truncate sequence in DADA2?

Hello everyone,

I am quite new to qiime2 but I am eager to learn how to use it. I am having trouble to decide were to truncate the sequence in DADA2.

I got my demultiplexed paired end sequences and the length of my forward primer (ITS9) is 23 bp and of reverse primer (ITS4) is 20. The amplicon lenght of should be between 240-460bp. I have also uploaded the interactive plot visualization. The sequence quality of the forward reads looks good but the quality of the reverse reads is lower and I don’t know where to truncate. It would be great if you all can help me in understanding and deciding where to truncate my sequences.


Here my command in QIIME2

qiime dada2 denoise-paired
–i-demultiplexed-seqs demux-paired-end.qza
–p-trim-left-f 23
–p-trim-left-r 20
–p-trunc-len-f 240
–p-trunc-len-r 210
–o-table table-dada2.qza
–o-representative-sequences rep-seqs-dada2.qza
–o-denoising-stats stats-dada2.qza

Thank you in advance,
Davide!

Hi @Pikula,

I am not an expert on ITS, but I will try to share my little experience!
First of all, on which region the primers anneal? If ITS1 or ITS2 you can use ITSxpress to extract only the ITS region form the sequences: Q2-ITSxpress: A tutorial on a QIIME 2 plugin to trim ITS sequences

I would be a bit skeptical to apply a fixed length threshold across all the sequences given the high length variability you giving. I would be worried to loose many of the good longer ITS amplicons. Especially because, looking at the length of your sequences, if you have a max length of 460bp, you have barely an overlap between R1 and R2, so you may want to be fairly relaxed with the trimming to not loosing it.

I would therefore keep ‘–p-trunc-len-f 0’ and ‘–p-trunc-len-r 0’, this will leave on the default quality trimming applied to all the sequences (you may choose a stricter quality then the default if you like!). If you apply ITSxpress, it should behind the scene perform a quality trimming as well.

But again, this is just my thought, hope it helps.

Luca

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Hi @llenzi,
many thanks for your answer. Right now I am doing some tests changing the values for truncating the reverse sequences to check the differences in the output I get.
In the next days I will also check the ITSxpress tools since I am curious to see how it works.

Many thanks again for your suggestions,

Davide