Demux and visualization of data

[HebaHussein-1981 (Heba Hussein)]

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

[ben (benjamin w.)]

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

[HebaHussein-1981 (Heba Hussein)]

they range between 40-60 k

[ben (benjamin w.)]

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

[HebaHussein-1981 (Heba Hussein)]

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.

[ben (benjamin w.)]

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

[HebaHussein-1981 (Heba Hussein)]

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?

[HebaHussein-1981 (Heba Hussein)]

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)

[ben (benjamin w.)]

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

[HebaHussein-1981 (Heba Hussein)]

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?

[ben (benjamin w.)]

Yes, put them into one line.

Also, all the of – were converted into the long dash -. Ben

[HebaHussein-1981 (Heba Hussein)]

I got a message to add (f) to the trim and I tried adding f and still getting error. how we can make the trim zero to make it easier?
qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux.qza --p-trim-left-f-6 --p-trim-right-f --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

(1/2?) no such option: --p-trim-left-f-6 (Possible options: --p-trim-left-f,

–p-trim-left-r)

(2/2?) no such option: --p-trim-right-f

[ben (benjamin w.)]

qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux.qza --p-trim-left-f 6 --p-trim-left-r 7 --p-trunc-len-f 225 --p-trunc-len-r 218 --p-n-threads 0 --o-table table.qza --o-representative-sequences rep-seqs.qza --o-denoising-stats denoising_stats.qza

--p-trim-left-f-6 (in case you wanted to identify the issue, there was a dash that connected your modifier "--p-trim-left-f" to "-6" and qiime didn't like it!

Not sure why your code has dashes all over the place, but that's ok. Try running that above. Ben

[HebaHussein-1981 (Heba Hussein)]

thanks it seems running but it seems will take long time. although I am using only 6 sample. one tutorial i watched said it took 4 days. also, I got a message that the path incorrect so I entered the aboslute path although in all tutorials they use relative path

[ben (benjamin w.)]

Depends on how complex the samples are (richness/diversity). If they are stool, it may take a while, but I would recommend running it on a beefy computer (+ a lot of RAM / decent processor). Ben

[HebaHussein-1981 (Heba Hussein)]

thanks it run but cant find the files on desktop as usual and when I searched on mac, cant reach them. also, visualization of these three file using the same website as we did before?!

edit: I will try to summarize it as I did for the visualization to see it
DADA2|690x61

[ben (benjamin w.)]

@ben

qiime dada2 denoise-paired --i-demultiplexed-seqs /Users/hebahussein/Desktop/paired-end-demux.qza --p-trim-left-f 6 --p-trim-left-r 7 --p-trunc-len-f 225 --p-trunc-len-r 218 --p-n-threads 0 --o-table /Users/hebahussein/Desktop/table.qza --o-representative-sequences /Users/hebahussein/Desktop/rep-seqs.qza --o-denoising-stats /Users/hebahussein/Desktop/denoising_stats.qza

You should output your files to a place where you know you can find them, such as a desktop fold. Ben

[thermokarst]

Yep, this is great advice! Just to follow up though, @HebaHussein-1981, your files were output to your Home Directory, you can find them there for now.