About reverse primer removal

[Beatriz Gil Pulido]

Hi, 

I would like to ask about the reverse primers sequence in the mapping file. Following the qiime pipeline, it is recommended to remove it from the mapping file during the demultiplex step. What about to work with a mapping file from the denoising step (since the start) without specifying the reverse primers? So just a mapping file with the name of each sample, the barcode sequence, the primer sequence (not the reverse one) and the description column. 

Regards, 

[Jai Ram Rideout]

Hi Beatriz,

Take a look at the following threads for info about denoising and reverse primers:

https://groups.google.com/d/topic/qiime-forum/0Q5rxsTxP3A/discussion

https://groups.google.com/d/topic/qiime-forum/227uUfpZOfI/discussion

https://groups.google.com/d/topic/qiime-forum/RMPhxpzflXI/discussion

https://groups.google.com/d/topic/qiime-forum/FRxiDlBJ3Xw/discussion

Best,

Jai

[Beatriz Gil Pulido]

Hi Jai, 

Thanks for your answer and for recommended links. But I´m still confused with that question. I´ve already performed all my analysis but I want to be sure I did all the process in the correct way. 

In the threads they talked about denoising using qiime. In my case, I did the denoising step using Acacia prior the split library. Once I ran the split_libraries command I used the mapping file without to include the reverse primer. I used the barcode and the forward primer. And that mapping file is the one I´ve used for the other commands used. 

I don´t really think it makes any difference, in my case, the mapping file with or without specifying the reverse primer sequence, but I just want to be sure about that question. 

[Jai Ram Rideout]

Hi Beatriz,

I've contacted a couple developers that should be able to help you with your question.

Best,

Jai

[TonyWalters]

From what I've read, Acacia only generates a fasta file output, so one would be limited in using Acacia before the split_libraries.py step (as you could not make use of the .qual files). We certainly don't have a "correct" approach to integrating Acacia-based results, but I would probably go this route:

run split_libraries.py (and use the reverse primer removal options at this step).
Run acacia on the resulting seqs.fna file, making sure not to use the split_on_mid option. I'm not sure if it will alter the fasta labels in the output though, which could complicate things, and the user will need to check that the resulting output fasta file looks good with the http://qiime.org/scripts/validate_demultiplexed_fasta.html script.

[Beatriz Gil Pulido]

Thanks Jai, 

Bea. 

[Beatriz Gil Pulido]

Hi Tony, 

Thanks for your answer. 

I ran acacia before the split library (avoiding the split_on_mid option) with both .fasta .qual files. Then I ran the split library with the generated output. I set the -z option in  the split library script and I checked the differences with the output generated without removing the reverse primers. There is no difference at all in the split_library output but there is in the following steps. Differences are not large but  I think is more accurate to use that filter too and the rarefaction and diversity index look better. 

Regards, 

Bea.