The next step in the tutorial is demultiplexing the sequences.
The code given in the tutorial:
qiime demux em-single \
--i-seqs emp/sequences.qza \
--m-barcodes-file sample-metadata.tsv \
--m-barcodes-column barcode-sequence \
--o-per-sample-sequences demux.qza \
Didn’t work, although I was in the moving-pictures folder and sequences.qza in the emp-subfolder.
(1/2) Missing option ‘–o-error-correction-details’. ("–output-dir" may also be used)
(2/2) Got unexpected extra arguments (o-error-correction-details demux-details.qza)
My solution was to change
--o-error-correction-details demux-details.qza in
And it worked. demux.qza and demux-details.qza were both saved in my moving-pictures folder, although I didn’t type in the whole path for demux.qza.
Is there anybody out there who can explain to me, why that is?
About the demux-details.qza the tutorial says "you can visualize these data using
qiime metadata tabulate". I am not happy to say: no, you can’t. I tried it.
qiime metadata tabulate demux-details.qza
(1/3) Missing option ‘–m-input-file’.
(2/3) Missing option ‘–o-visualization’. ("–output-dir" may also be used)
(3/3) Got unexpected extra argument (demux-details.qza)
Oh, ok. Interesting.
Next step in the tutorial is to visualize the data.
qiime demux summarize \
--i-data demux.qza \
It worked!!! and made me so happy … until I tried to see the results by
qiime tools view demux.qzv
Bad decision to run that code. Bad bad decision. A new window in Safari appeared with all the information about the dataset. I closed the tab and returned to the terminal only to face the fact, that something is wrong again:
Press the 'q' key, Control-C, or Control-D to quit. This view may no longer be accessible or work correctly after quitting.
Control-D and Control-C didn’t work, so I had to quit the whole thing.
Note to myself: never try to see your results ever again …
Based on the plots you see in demux.qzv, what values would you choose for --p-trunc-len and --p-trim-left in this case?” I don’t dare to plot the dataset again, so no, thanks.
But I wonder how I ever will be able to work properly with my own data when I can’t visualize it?!
--i-demultiplexed-seqs demux.qza \
--p-trim-left 0 \
--p-trunc-len 120 \
--o-representative-sequences rep-seqs-dada2.qza \
--o-table table-dada2.qza \
New insight: o = output and i = input.
Question: what is p and len? My guess: p = please, len = Lenny. please trim 0 at the left, please truncate Lenny at 120.
qiime metadata tabulate \
--m-input-file stats-dada2.qza \
Worked nicely. What means the m? My guess: m = my. My input file stats-dada2.qza.
Good news. I did the dada2 and deblur part without any difficulties.
I only would like to add, that instead of
mv rep-seqs-deblur.qza rep-seqs.qza
mv table-deblur.qza table.qza
It is possible to just go to the finder and rename the files.
Fortunately I will to all further analyses in R, so after I figured out, that my data is already in Casava 1.8 demultiplexed format, I will try the corresponding import data tutorial now.
Wish me luck!