Dear All, we have some 16S and ITS data set after the data2 denoising the merge rate is very low. The below is the detail. Can you please give me some pointers about what is the reason and how should I troubleshoot? Appreciate it in advance.
Hey there @archie - you forgot to include the command or commands you ran - we will need that to contextualize your other attachments. Thanks!
Thank, the CMD is below
nohup qiime dada2 denoise-paired \
--i-demultiplexed-seqs demux-paired-end.qza \
--p-trim-left-f 7 \
--p-trim-left-r 7 \
--p-trunc-len-f 270 \
--p-trunc-len-r 220 \
--p-n-threads 55 \
--o-representative-sequences rep-seqs.qza \
--o-table table.qza \
--o-denoising-stats stats.qza \
>denoising.log 2>denoising.err &Wild guess:
Your amplicon might be too long to be merged after the truncation (–p-trunc-len-f & --p-trunc-len-r) of your reads.
After truncation there should be a remaining overlap of >20bp in order to merge the reads, maybe this isn’t the case? How long is your amplicon supposed to be? Be aware of natural length variation!
Solution if the above is true: choose larger values for --p-trunc-len-f and/or --p-trunc-len-r.
Let me try with a less truncation, Thanks,
For the ITS, that might be the issue. The insert is 600bp and we run Miseq PE300. Is that possiable to fix this?
No — there is no way to join paired sequences if they do not overlap.
ITS is hypervariable in length, so 600 is probably just an estimate (median length?). So you could give it a try and see how many reads join... though that would make me a little anxious personally, since the excluded reads (those that fail to join) will come from specific taxonomic groups, so you would effectively be skewing the taxonomic profiles.
A better bet (and perhaps your only choice) would be to use only the forward ITS reads and process as single-end data.
If you do still want to try paired ITS, at least run the single-end and compare the taxonomic profiles to make sure that read join failures are not skewing your taxonomy profiles.
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