reverse primer removal in denoising or not?

[Vanessa V.]

Hi all,

I think I am getting confused as to what step the reverse primer (at
the 3` end) needs to be removed. I know that it definitely needs to
be removed prior to OTU picking. However, I seem to be finding
conflicting posts whether the reverse primer has to be left in prior
to denoise_wrapper or has to be removed prior to denoise_wrapper. Can
anyone let me know what is the correct way (before or after
denoising), just want to make sure I am performing the denoiser step
correctly.

Thanks,
Vanessa

[Jose Carlos Clemente]

Hi Vanessa,

the forward and reverse primer should be removed during
split_libraries, specify it in the mapping file and then use the -z
option with split_libraries.py

Jose

[Vanessa V.]

OK, so removing the primers at this step will remove and won't affect
the .sff.text file used for the flow gram clustering (denoising)?
Sorry, I am getting absolutely confused, because someone else on the
forum had said the primers cannot be removed prior to denoising. Am I
missing something?

On May 7, 12:18 pm, Jose Carlos Clemente <jose.cleme...@gmail.com>
wrote:

[Tony Walters]

Hello Vanessa,

You would want to first denoise the sequences, the remove the reverse primer sequences (with truncate_reverse_primers.py), then do OTU picking and the downstream steps from there.

-Tony

[Vanessa V.]

OK thanks Tony. Sorry I was getting so confused!

[Vanessa V.]

Also, where can I find the parameters for truncate_reverse_primers.py?

[Tony Walters]

Oopos, it's actually truncate_reverse_primer.py

You want to pass the .fna output file of denoising with -f, your mapping file with -m, and an output directory with -o 

You should be able to see these with truncate_reverse_primer.py -h

I would note that this is in the development version of QIIME.  It was discussed on a previous thread, but I wasn't sure if you manually installed the code or not.

-Tony

[Vanessa V.]

I wasn't quite sure how to manually install it - I have downloaded a
newer version of QIIME recently, so I was thinking I could go ahead
and just use truncate_reverse_primer.py. Can that not be done?

[Tony Walters]

If you downloaded the svn version of QIIME, yes.  If it was the 1.4.0 release version of QIIME, no.

The stand-alone version of the truncate_reverse_primer.py code are attached/described in this thread (5th message from the bottom):

http://groups.google.com/group/qiime-forum/browse_thread/thread/ef1e474cdfbf3fce/

Configuring these files depends upon how you have QIIME installed.  If it's macqiime, you'd want to follow the last message in the thread.  If it's a native installation, you would have to copy the library (/qiime/) file into your library folder (could be /qiime/ or a /lib folder) locally and the script into the /scripts/ (or /bin/) folder.

Alternatively, as we are planning on releasing QIIME 1.5.0 today, you could wait and just install the next release.

-Tony

[Vanessa V.]

Hi Tony,

Thanks!

Can you let me know where I can find QIIME version 1.5.0?

On May 7, 1:08 pm, Tony Walters <william.a.walt...@gmail.com> wrote:
> If you downloaded the svn version of QIIME, yes.  If it was the 1.4.0
> release version of QIIME, no.
>
> The stand-alone version of the truncate_reverse_primer.py code are

> attached/described in this thread (5th message from the bottom):http://groups.google.com/group/qiime-forum/browse_thread/thread/ef1e4...