Demux and visualization of data

Hi Ben. I am trying to summarize (visualize) the file. I had an error related to output path:
What I used:qiime demux summarize
–i-data /Users/hebahussein/Desktop/paired-end-demux.qza
\
–o-visualization /Users/hebahussein/Desktop/paired-end-demux.qzv

the error: (1/1) Missing option “–o-visualization”. ("–output-dir" may also be used)

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qiime demux summarize --i-data /Users/hebahussein/Desktop/paired-end-demux.qza --o-visualization /Users/hebahussein/Desktop/paired-end-demux.qzv

Try running this command. Ben

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thanks it worked

Saved Visualization to: /Users/hebahussein/Desktop/paired-end-demux.qzv

it seems that it is better to be continuous rather than separated by backslash

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Yes, these “” tells the command to go to the next line. You can do away with all backslashes and run it in one line. It just helps visualize the commands. Ben

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so, I try to view the output file. I tried both the absolute and relative path and both got error(qiime2-2019.7) a-PC:~ hebahussein$ qiime tools view/Users/hebahussein/Desktop/paired-end-demux.qzv
Usage: qiime tools [OPTIONS] COMMAND [ARGS]…
Try “qiime tools --help” for help.

Error: No such command “view/Users/hebahussein/Desktop/paired-end-demux.qzv”.
(qiime2-2019.7) a-PC:~ hebahussein$ qiime tools view paired-end-demux.qzv
Usage: qiime tools view [OPTIONS] VISUALIZATION

Displays a QIIME 2 Visualization until the command exits. To open a QIIME
2 Visualization so it can be used after the command exits, use ‘qiime
tools extract’.

Options:
–index-extension TEXT The extension of the index file that should be
opened. [default: html]
–help Show this message and exit.

                There was a problem with the command:                     

(1/1) Invalid value for “VISUALIZATION”: File “paired-end-demux.qzv” does not
exist.
(qiime2-2019.7) a-PC:~ hebahussein$

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go too view.qiime2.org

drag this file into the window.

Ben

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very cool. but is this output seems normal? also, I pressed download both as pdf and file but did not find it in download

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Not sure, it looks ok, but I agree odd. Can you tell me how many samples you had again? Ben

edit: you had six.

can you click around and tell me how many forward/reverse sequences were demultiplexed per sample?

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this is the interactive view
but still I cant download the file or interpretive the results
edit: I am not sure though if this is visualization of my data or saved on the website. i just choose interactive tab after dragging my qzv

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Hm, what do you want to download? It looks otherwise pretty good! Ben

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thanks a lot. I need to download the visualization as a pdf.
edit: I downloaded sequence count as pdf. thanks

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I think there should be an option, but I think your frequency and quality graphs look good. You can proceed with DADA2 if you’re ready. As for the visualizations, I would click around the page.

On the first page, you can scroll ALL the way down to download the CSV that tells you how many sequences per sample (which is important). You can plot them using another program such as graphpad (prism).

Ben

edit: show me the sequence counts please

edit.edit: I think these may be V3-V4 16S, do you know what kind of 16S region that these were? Ben

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they range between 40-60 k

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Good enough, you can send it through DADA2 now. Ben

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thanks, I am trying. how to know where to trim? what is the accepted threshold? also I have paired-end so how to make sure they overlap? Finally, how long may it take to run as I read it may take 4 days? I only trying on 6 samples not all my samples.

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forward should be around 200
reverse should be 160-180

You should look at your quality plots and see where they start to drop. You want to trim before the quality drops. Ben

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as I understand, we put the cutoffs after trunc command. which numbers should be after the trim please?qiime dada2 denoise-paired
–i-demultiplexed-seqs paired-end-demux.qza
–p-trim-left 6
–p-trim-left-r7
–p-trunc-len-f 225
–p-trunc-len-r 218
–o-table table.qza
–o-representative-sequences rep-seqs.qza
–o-denoising-stats denoising_stats.qza

edit: which video tutorial topics should I watch to understand quality score and the plots?

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so, Ben which numbers should we choose for trim?
based upon what?
any tutorial to help me understand and decide?
thanks
edit: because I keep on getting same error (1/2?) no such option: --p-trim-left (Possible options: --p-trim-left-f,

–p-trim-left-r)

(2/2?) no such option: --p-trim-left-r7 (Possible options: --p-trim-left-f,

–p-trim-left-r)

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Trim #s should be based on if you have extra NT that you think may have been left from your primers. Here your trim # can be zero - I sometimes out of habit will trim 8-13 off of the forward and reverse. This should not impact your DADA2 results. Ben

qiime dada2 denoise-paired
–i-demultiplexed-seqs paired-end-demux.qza
–p-trim-left 6
–p-trim-right 7
–p-trunc-len-f 225
–p-trunc-len-r 218
–o-table table.qza
–o-representative-sequences rep-seqs.qza
–o-denoising-stats denoising_stats.qza

–p-trim-left-r7

This is a typo, please be careful w/ your commands! Ben

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thanks i got many errors so i need to try all command continuous without backslash. so u cut and paste or re-type the command?

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