Hi, all! I'm a beginner learning metagenome analysis using qiime2.
It's quite tough to learn it by myself :(
I have 3 questions in total about specific stage in analysis process using qiime2.
I read a manual in qiime2 homepagedocs.qiime2.org) about training feature classifier, and there is one thing I don't get it. They say 'Taxonomic classifiers perform best when they are trained based on your specific sample preparation and sequencing parameters. You should follow the instructions in Training feature classifiers with q2-feature-classifier to train your own taxonomic classifiers'. But as I understand, training classifier is in order to classify OTU I picked from my samples, which means they should be classified by trained classifier with reference database, SILVA (what I use) or Greengenes. Then what does that 'your specific sample preparation and sequencing parameters' mean?? What I used for training classifier is 'silva_132_99_16S.fna'(I import it with using qiime tools import) in rep_set directory of SILVA_132_QIIME_release for reference reads input data and 'taxonomy_all_levels.txt' for reference-taxonomy input data.
With using the classifier (in my former question), I did taxonomic analysis my samples. I tried 9 samples and I got open reference otu clusters by following clustering processes in 'docs.qiime2.org'. I made a bar plot afterwards, and it shows only 6 samples analysis data, without 3 samples'. Is this a problem of my classifier? or metadata of samples or something?? Or does this mean there are no reference sequenced bacterial samples matched in those 3 samples?
After I denoise these samples, I tried to do 'qiime diversity core-metrics-phylogenetic' function. These are my Per-sample sequence counts.
sample-1 : 20237 sample-2 : 11372 sample-3 : 10039 sample-4 : 9269 sample-5 : 8563 sample-6 : 6431 sample-7 : 4939 sample-8 : 4095 sample-9 : 3105
I tried it with --p-sampling-depth 3100 at first, it says 'Plugin error from denoise(name of directory I did this function).The rarefied table contains no samples or features. Verify your table is valid and that you provided a shallow enough sampling depth.' So, I raised depth 5000, 8000 and 18000 but it reply the error every time. Is this a matter with the metadata? And which value I choose for this function? As I understand, the sampling depth means the amount of subsample in each sample, so if I input 4000 for sampling depth, qiime picks 5000 subsample for doing diversity analysis, so qiime picks all samples in sample 7, 8, and 9 while it picks only 5000 out of each total samples. Which means in case of sample 1, they are subsampled only a quarter of them, which could be slightly precise for it.