before and after trimming, difference between qzv files

[Parul_Baranwal]

Hello everyone,

I am running 26 paired end sequences (16s RNA) which is sequenced through illumina platform PE 250.
I demuliplexed the data and the demultiplexed files is attached below:
demux-paired-end.qzv (288.4 KB)

I have following questions:
I trimmed both forward and reverse sequences and the trimmed file is as follow:
trimmed-remove-primers.qzv (293.9 KB)

1. When I am opening both qzv files, I observed for the forward reads, the quality drops a lot for the sequences which are greater than 220 bases. For the reverse reads, the bases are cut after 225 bases and the quality is still low.
   As far as I understood, after trimming step, quality should increase but its decreasing. Can someone please help me to understand.
2. Based on the trimmed-remove-primers.qzv file, I am using the following code for dada2 processing but its running for hours. I am not sure if its okey. I am using virtual machine and I have alloted 20000 MB memory space to VM.
   
   

Thank you in advance!

[thermokarst]

Hi @Parul_Baranwal!

@Parul_Baranwal

As far as I understood, after trimming step, quality should increase but its decreasing. Can someone please help me to understand.

By looking at provenance - I noticed you didn't trim anything off of the 3' end, only the 5' end, so nothing is going to change with respect to the "right" side of the plots. Second - the qiime demux summarize plot uses a random subsample (controllable with the --p-n parameter) - you are seeing a slightly different subsampling between plots, too.

@Parul_Baranwal

I am not sure if its okey

Those parameters look good to me, that is also where I would choose to truncate at, on my first pass through these data.

Give it time! Also, it looks like you didn't specify multiple threads - this will run significantly faster with more threads. Check out the --help text for more info!