I have some questions about sequence quality. In my case, demux.qzv from paired-end read data is shown below.
It seems to me that QIIME2 tries to get a random sampling of 10000 sequences. I noticed that a previous discussion on QIIME2 quality score suggested a cut-off value of 20 for score. For my data, the amplicon length is about 250 nts. I noticed from the Interactive Quality Plot that number of sequences used drops dramatically from 10000 to only around 1000 when the length is greater than 240.
Question 1: For the down stream analysis by denoting with DADA2, is it reasonable to set length at 240 nts and use the following command (no trimming of the left side)? Should I also truncate the left side, e.g. -p-trim-left-f 10 according to the plot?
qiime dada2 denoise-paired --i-demultiplexed-seqs demux-paired-end.qza --p-trim-left-f 0 --p-trim-left-r 0 --p-trunc-len-f 240 --p-trunc-len-r 240 --o-table table.qza --o-representative-sequences rep-seqs.qza --o-denoising-stats denoising-stats.qza
The demultiplexed sequence counts summary is shown below.
Question 2: For "qiime diversity core-metrics-phylogenetic" command group (qiime diversity core-metrics-phylogenetic --i-phylogeny rooted-tree.qza --i-table table.qza --p-sampling-depth ? --m-metadata-file MetadataT.txt --output-dir core-metrics-results) , what number is proper as the parameter for sampling depth " --p-sampling-depth". Base on the second plot attached, should I use the minimum sequence account, i.e. 141860?
Question 3: For the "qiime diversity alpha-rarefaction" command group (qiime diversity alpha-rarefaction --i-table table.qza --i-phylogeny rooted-tree.qza --p-max-depth ? --m-metadata-file MetadataT.txt --o-visualization alpha-rarefaction.qzv), what number is proper as the parameter for max depth " --p-max-depth". Base on the second plot attached, should I use a number between the minimum sequence account, i.e. 141860, and median, i.e. 178898?
Thanks a lot in advance!