Dada2 trunc-len 0 for ITS analyses

Hi,

I am analyzing fungal ITS2 (ITS86F/ITS4 primers, PE300) sequences and I have some questions regarding the truncation of the reads as well as dealing with amplicons of different lengths.
In several sites I have seen that it's advised to put the truncation length at dada2 denoise step to "0".
(https://github.com/Joseph7e/ITS_metabarcoding_analyses
DADA2 Pipeline Tutorial (1.16) )
This makes sense as ITS has very variable length depending on the species - using a specific truncation length for all reads will bias the results (as you loose all the shorter amplicons). At the same time when setting the truncation length to 0 I have a lot less sequences that pass the merging step. I tried it as well on data that has been passed through cutadapt.
I found some explanation at this thread DADA2, truncation lengths and features number - but in the end I still don't know how to proceed with my data so that I could keep all quality reads... Maybe ITSxpress will help (posted another topic on my issues with that tool).

demux-paired-SB72.qzv (270.0 KB)
trimmed_sequencesSB72.qzv (274.4 KB)

Original data:

qiime dada2 denoise-paired
--verbose
--i-demultiplexed-seqs demux-paired-end_SB72.qza
--p-trim-left-f 0
--p-trim-left-r 0
--p-trunc-len-f 270
--p-trunc-len-r 220
--p-n-threads 40
--o-table table_0SB72.qza
--o-representative-sequences rep-seqs_SB72.qza
--o-denoising-stats denoising-stats_truncSB72.qzv

denoising stats
sample-id input filtered denoised merged non-chimeric
SB72 18647 14620 14620 14513 14466

qiime dada2 denoise-paired
--verbose
--i-demultiplexed-seqs demux-paired-end_SB72.qza
--p-trim-left-f 0
--p-trim-left-r 0
--p-trunc-len-f 0
--p-trunc-len-r 0
--p-n-threads 40
--o-table table_0SB72.qza
--o-representative-sequences rep-seqs_0SB72.qza
--o-denoising-stats denoising-stats_0SB72.qza

denoising stats
sample-id input filtered denoised merged non-chimeric
SB72 18647 11091 11091 298 298

The cutadapt trimmed data - removed the part that for short amplicons runs into reverse primer

qiime dada2 denoise-paired
--verbose
--i-demultiplexed-seqs trimmed_sequences.qza
--p-trim-left-f 0
--p-trim-left-r 0
--p-trunc-len-f 270
--p-trunc-len-r 220
--p-n-threads 40
--o-table table_trimmedSB72.qza
--o-representative-sequences rep-seqs_trimmedSB72.qza
--o-denoising-stats denoising-stats_trimmedSB72.qza

denoising stats
sample-id input filtered denoised merged non-chimeric
SB72 18647 13822 13822 13770 13770

qiime dada2 denoise-paired
--verbose
--i-demultiplexed-seqs trimmed_sequences.qza
--p-trim-left-f 0
--p-trim-left-r 0
--p-trunc-len-f 0
--p-trunc-len-r 0
--p-n-threads 40
--o-table table_0trimmedSB72.qza
--o-representative-sequences rep-seqs_0trimmedSB72.qza
--o-denoising-stats denoising-stats_0trimmedSB72.qza

denoising stats
sample-id input filtered denoised merged non-chimeric
SB72 18647 11488 11488 703 703

So in the end I am having trouble with the logical recommendation for ITS not to truncate the reads, but then loosing most of my reads.

Thank you in advance!!!

I think as you noted above, this is because of read-through through the primer at the 3’ ends of the reads. So untrimmed reads leave the read-through, which cannot align :disappointed:

Are you sure cutadapt is removing everything? The reverse primers on the forward reads and the forward primers on the reverse reads? From those demux summaries it looks like only a fraction of sequences were actually trimmed, which seems surprising, though with ITS it’s always questionable what the expected read length is if you don’t know what species are present… :mushroom: :sob: :mushroom:

q2-ITSxpress should take care of the trimming issues :slight_smile:

But otherwise it looks like the trimmed reads are working quite well for you — unless if there’s something wrong with that protocol that I’m missing, I’d say run with those if q2-ITSxpress does not fix things for you…

I hope that helps!

Hi,
I’m rather sure that cutadapt removes everything - only fraction was trimmed just because many of the amplicons were very long - so they didn’t have the read-through issue.
But finally I agree that the ITSxpress is the way to go :slight_smile:
Thanks!

1 Like

This topic was automatically closed 31 days after the last reply. New replies are no longer allowed.