Dear all,
I used 80 samples to detect the bacteria on the human skin.
the problem is that after I tried many sample depth there was no reliable curves to choose the proper sample depth. my question is that since there are some commands in qiime2 for alpha and beta diversity metrics which don't include the sample depth, is it ok if I use them? if yes, are they include all the sample reads when we don't choose any sample depth in the commands?
I share the commands I used and the pictures of the outputs.
Thank you.
To create a phylogenetic tree artifact.
qiime phylogeny align-to-tree-mafft-fasttree \
--i-sequences rep-seqs.qza \
--o-alignment aligned-rep-seqs.qza \
--o-masked-alignment masked-aligned-rep-seqs.qza \
--o-tree unrooted-tree.qza \
--o-rooted-tree rooted-tree.qza
Then create a “alpha diversity” folder.
mkdir alpha-diversity
mkdir alpha-diversity/alpha-rarefaction alpha-diversity/alpha-diversity-index alpha-diversity/alpha-group-significance
Then create the rarefaction curve.
qiime diversity alpha-rarefaction \
--i-table table.qza \
--i-phylogeny rooted-tree.qza \
--p-metrics chao1 \
--p-metrics observed_features \
--p-metrics shannon \
--p-metrics faith_pd \
--p-max-depth 6500 \
--m-metadata-file sample-metadata.txt \
--o-visualization alpha-diversity/alpha-rarefaction/sample_depth-6500.qzv
qiime dada2 denoise-paired \
--i-demultiplexed-seqs paired-end-demux.qza \
--p-trim-left-f 30 \
--p-trim-left-r 30 \
--p-trunc-len-f 240 \
--p-trunc-len-r 220 \
--o-representative-sequences rep-seqs-dada2.qza \
--o-table table-dada2.qza \
--o-denoising-stats stats-dada2.qza \
--verbose
To check the alpha diversity value.
qiime diversity alpha \
--i-table table.qza \
--p-metric shannon \
--o-alpha-diversity alpha-diversity/shannon-SampleData[AlphaDiversity]
To get the diversity index.
qiime metadata tabulate \
--m-input-file alpha-diversity/shannon-SampleData[AlphaDiversity].qza \
--o-visualization alpha-diversity/alpha-diversity-index/shannon-index
To perform the statistical analysis.
qiime diversity alpha-group-significance \
--i-alpha-diversity alpha-diversity/shannon-SampleData[AlphaDiversity].qza \
--m-metadata-file sample-metadata.txt \
--o-visualization alpha-diversity/alpha-group-significance/shannon-significance.qzv