hello, can anyone help me in dada2 denoising step in qiime 2. i am not able to merge my sequences. which trunc length to choose?
/home/qiime2/Screenshot from 2021-08-21 15-50-32.p
Hello @ANKITA1,
I wrote up a guide about choosing merging settings, which you might find helpful.
If you have any questions, let me know!
Colin
total length of Amplicon is 465 bp , V3-V4 region
can any only help me in choosing the trunc. lenth of forward and reverse reads as i tried many trunc parameter and i am getting maximu 30% merging7 off-topic replies have been split into a new topic: I wonder what you suggest when sequence quality drops off markedly before the R1 and R2 reads will overlap
Please keep replies on-topic in the future.
I also encountered similar situation when I was trying to analyze our old data and new data at the same time. Their lengths and quality score are quite different. Then I found that running deblur workflow is much easier.
qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux.qza --p-trunc-len-f 250 --p-trunc-len-r 240 --p-trim-left-f 20 --p-trim-left-r 19 --p-trunc-q 2 --o-representative-sequences rep-seqs-dada2.qza --o-table table-dada2.qza --output-dir dada2-dmx-pe_data.qzv
This command i run and i got only 34% merging. will that be ok to further process and analyse . as most of the reads got filtered out
Thank you for sharing that information.
With that data, we can do the math:
No truncation: 250 + 250 - 465 = 35 bp overlap
With --p-trunc-len-f 245 --p-trunc-len-r 240
: 245+240-465=20 bp overlap.
These overlap values are both long enough, but more reads will probably join if you truncate off the low quality bases at the end.
What --p-trim-left-f
and --p-trim-left-r
do you plan to use?
Hello again
Sorry for my delay. Thanks for being patient.
Those settings make sense to me, and give you plenty of overlap so your reads can merge!
That's not good, but I think I know why it's happening...
The dada2 plugin for Qiime2 merges paired-end reads using the defaults of dada2 (link to code), which is maxMismatch = 0
for this function. So any differences in the area of overlap will cause your reads not to join, leading to low values like 34%.
To fix this, try truncating of more basepairs from the ends of the reads.
This topic was automatically closed 31 days after the last reply. New replies are no longer allowed.