cutadapt and dada2

[zhaohuicheng (zhao)]

Dear all!
When I use dada2 to denoise my data,In the first, I use the cutadapt to trim the primer.
Then using the dada2 to denosie my data. ** I found nearly all the sequence were trimmed.
when I try to use the dada2 only,just change the number about -p-trim-left-r , the outcome data is ok.
I want to trim the primer first, and then denoise the data. What can I do?

qiime cutadapt trim-paired
–i-demultiplexed-sequences paired-end-demux_B.qza
–p-cores 4
–p-front-f GTGYCAGCMGCCGCGGTAA
–p-front-r GGACTACNVGGGTWTCTAAT
–o-trimmed-sequences trimmed-seqs_B.qza
–verbose

qiime dada2 denoise-paired --i-demultiplexed-seqs trimmed-seqs_B.qza --p-trim-left-r 0 --p-trim-left-f 0 --p-trunc-len-f 300 --p-trunc-len-r 260 --o-representative-sequences rep-seqs_B.qza --o-table table_B.qza --o-denoising-stats stats_B.qza --p-n-threads 0 --verbose

[Nicholas_Bokulich]

Hi @zhaohuicheng,
dada2 has 5’ trimming (e.g., to remove primers) built in. --p-trim-left-f and --p-trim-left-r will do it for you, so there is no need to use q2-cutadapt first.

Unless, of course, if your primers are not flush with the 5’ end start at variable locations. In that case cutadapt should be fine, but just make sure you re-check the seq lengths (with qiime demux summarize) to make sure your trunc parameters are appropriate (anything shorter than the trunc length is discarded).

I hope that helps!

[zhaohuicheng (zhao)]

Thanks for your reply. Because my primer is GTGYCAGCMGCCGCGGTAA it were add 1-5 random bases, like this:
|1|PFV4| GTGYCAGCMGCCGCGGTAA|
|2|PFV4-1| TGTGYCAGCMGCCGCGGTAA|
|3|PFV4-2| CTGTGYCAGCMGCCGCGGTAA|
|4|PFV4-3| ACTGTGYCAGCMGCCGCGGTAA|
|5|PFV4-4| GACTGTGYCAGCMGCCGCGGTAA|
|6|PFV4-5|AGACTGTGYCAGCMGCCGCGGTAA|
if I use the --p-trim-left-f and --p-trim-left-r parameters,It will loss 1-5 useful bases. So can I sacrifice 1-5 bases? Just use dada2 to do the next steps?thanks you!

[zhaohuicheng (zhao)]

Many thanks, I will try to change the length parameters.