Can anyone advise me about this issue? After running the command below
qiime dada2 denoise-single --i-demultiplexed-seqs qm/demux.qza --p-n-threads 64 --p-trim-left 0 --p-trunc-len 0 --o-representative-sequences qm/rep-seqs.qza --o-table qm/table.qza --o-denoising-stats qm/dada2-stats.qza --verbose
I got only few denoised sequences of some samples. Would it be possible to adjust the command to get more sequences?
Can you upload a picture of the interactive quality plot in your demux.qzv? I think that more conservative truncation values might fix this, but I will need to see the interactive quality plot to confirm.
These sequences were ccs generated by Pacbio sequel II. Before importing qiime2, I had removed the barcode and primer sequence, and filtered out reads smaller than 1200 or larger than 1800.
I have also tried using this command to denoise and obtained similar results as before.
qiime dada2 denoise-ccs --i-demultiplexed-seqs demux.qza --p-n-threads 64 --o-representative-sequences rep-seqs.qza --o-table table.qza --o-denoising-stats dada2-stats.qza --verbose --p-front ********* --p-adapter ********* --p-min-len 1200 --p-max-len 1800
The first sample seems to have been removed a lot of reads in the deonised step. I have another batch of samples, all of which show that a lot of reads have been discarded in the deonised step (denoise-ccs). Is there any command or parameter that can improve this situation?
The denoise-ccs command will remove the primers and adapters of reads and filter reads based on their max and min lengths. I want to obtain the clean data after removing primers and adapters, and filtering some reads based on length parameters.Clean data is very important to me. Could you tell me how to get these clean data.Thanks! demux.qzv (370.3 KB)
Yes I agree. One of your samples has a lot of reads removed due to denoising. However, sometimes samples just have bad quality and there is nothing that can really be done. My rule of thumb is if all of my samples lost 50% or more to denoising, then I am worried something went wrong.
If you want to mess around parameters there are a couple of parameters that you could test out.
You could try trimming at a higher value, right now it seems like you are not trimming at all. Trimming before your interactive quality plot starts dropping.
You could increase max expected error parameter: --p-max-ee. Just a warning though as you increase the max-expected-error threshold you will get more errors in your sequences.
The rep-seqs that you get out of denoised-ccs should be your clean reads.
Thanks for your advise. The cleandata I want is generated through the intermediate process of the following command. This command can remove adapters, primers, and filter reads based on minimum and maximum lengths (1200-1800). Then it began to denoise the sequence.
“qiime dada2 denoise-ccs --i-demultiplexed-seqs demux.qza --p-n-threads 64 --o-representative-sequences rep-seqs.qza --o-table table.qza --o-denoising-stats dada2-stats.qza --verbose --p-front ********* --p-adapter ********* --p-min-len 1200 --p-max-len 1800”.
I also checked the command you mentioned.
"qiime cutadapt trim-single"
This command can remove adapters, primers, and filter reads based on the minimum length, but it seems that it cannot filter reads based on the maximum length of the library. Perhaps I did not use this command correctly. Could you give me additional advice. Thanks again.