Dada2 - few sequences merged

[AndreFC]

Dear All,

Can anyone advise me about this issue? after running the command below
qiime dada2 denoise-paired
–i-demultiplexed-seqs demux.qza
–p-trim-left-f bp of primer F
–p-trim-left-r bp of primer R
–p-trunc-len-f length of best quality
–p-trunc-len-r length of best quality
–o-representative-sequences rep-seqs.qza
–o-table table.qza
–o-denoising-stats denoising_stats.qza
–p-n-reads-learn How many times it tries
–p-n-threads unlimited (0)
–verbose

I got only few merged sequences. would be possible to adjust the command to get more sequences? Ex the input was 29004 but only 8 sequences were merged.
See the attached file.

Andre

[sixvable]

Hi @AndreFC

Could you briefly describe your experimental design？Which amplicon you sequenced? Which sequencer and how long is the amplicon? Also upload your demux.qza so we can find what is the issue.

[wjschuiten (W Schuiten)]

Hey @AndreFC

Can you post your exact values for your trim and truncation parameters? These often cause the problem as there aren’t enough quality bases left to merge on.

Can you share your read quality as well? It helps determine optimal parameters. I got mine in R following the dada2 tutorial https://benjjneb.github.io/dada2/tutorial.html

[AndreFC]

Dear All,

In fact I tried to use short length of forward and reverse sequence (180, 210). When I increased that (240, 230) worked well.

–i-demultiplexed-seqs demux.qza
–p-trunc-len-f 240
–p-trunc-len-r 230