Hi All,
I have a question regarding the DADA2 denoising step. We sequenced the bacterial 16S rRNA genes in paired end mode (2 X 300 bp) (Illumina). The library was prepared using Qiagen kit, dual indexing approach. The sequencing platform had already finished the demultiplexing step. demux-paired-end-reads.qzv (317.2 KB) denoising-stats.qzv (1.2 MB) We don't know the primers information we used. So I performed the analysis on the demultiplexed data using QIIME2 starting from DADA2.
Here is the step and parameter; and I am attaching the demux qzv and the denoising-stats in this topic. Thank you
qiime dada2 denoise-paired
--i-demultiplexed-seqs ./demux-paired-end-reads.qza
--p-trim-left-f 0
--p-trim-left-r 0
--p-trunc-len-f 276
--p-trunc-len-r 276
--p-n-threads 20
--o-table ./table.qza
--o-representative-sequences /rep-seqs.qza
--o-denoising-stats ./denoising-stats.qza
Hi!
Here is a similar topic, check it out if it will help you to solve this issue
Hi Timanix
Thanks for your reply, I still got confused on the parameter set-up, would you please look into my output to see if you could provide some suggestions?
Thank you
Are primers already removed by sequencing center? If not, it is highly recommended to remove them by cutadapt before Dada2 since it may influent following taxonomy annotation.
As I wrote in another post by the link, you are loosing a lot of reads on the filtering step since you provided too high truncation values. Try to run it with lower values based on quality plots. Optimal values will be ones that truncate some bases with bad qualities at the end of the reads but keep enough overlapping region to merge paired reads. If you do not know amplicon size, you can try different values and choose best ones based on the stats.
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