Removal of primers and denoising issue!

[Hedvig]

Hi!

I am doing my first QIIME analysis on my Titanium amplicon run and I am concerned about a few things and I hope you can help me out. This is how I am doing:

1. First I run the split.library.py script with the default parameters (>200, <1000). (this is the command I am writing in the Terminal on my Mac: split_libraries.py -m Housing_map_R1.txt -f 1.TCA.454Reads.fna -q 1.TCA.454Reads.qual -o Housing_split_lib_R1). My question: This will remove forward primer, barcode and all sequences shorter than 200 and longer than 1000, right? I have used 16S primers 27F and 338R and I am not sure if it is proper to use the default settings or if I should change that?

2. Then I was thinking of running the same script again however also doing the trimming for the reverse primer (this is the script I ran: split_libraries.py -m Housing_map_R1_revP.txt -f 1.TCA.454Reads.fna -q 1.TCA.454Reads.qual -o Housing_split_lib_R1_revP --reverse_primer_mismatches 1 -z truncate_only). However, then I wanted to do denoising using the n3phele and as I have understood the denoiser program puts the reverse primer back. Is it so? I the n3phele using denoiser as denoise program? How should I deal with this? Should I just run the split.library.py without removing the reverse primer, then do the denoising and then deal with the reverse primer issue? Also, do I have to run the inflate script following denoising in n3phele or is it done there? And how do I deal with removing the reverse primer if I do it this way? Why I want to remove it is because I assume it will affect the OTU picking otherwise, or what is the proper way to do it?

3. Then I also had a question regarding trimming the sequences before OTU picking. Is that necessary, or does it deal with that the sequences will be of different lengths ( which I guess that they will be)?. If so, how do I decide where to trim them?

I hope you can help me solve this issues!

Best regards,

Hedvig

[Jose Carlos Clemente]

Hedvig,

concerning your first question, your setting looks correct except for
one thing: with Titanium, your reads will most probably include the
reverse primer and adapter, which you want to remove before OTU
picking (otherwise the clustering gets messed up because of the
adapter in the rev primer). So you do want to remove primers.

However, if you remove the reverse primer you cannot user
denoie_wrapper.py. Check this post (and link within) for further
information:

https://groups.google.com/forum/?fromgroups=#!topic/qiime-forum/lyKeKzxagnU

Finally, there is no need for you to trim sequences before OTU
picking. Unless you were to combine Titanium and FLX reads, in that
case you'd want to trim down the Titanium reads to FLX length
(otherwise the reads cluster by chemistry/length).

Jose

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[Hedvig]

Hi,

Thanks for you reply!

I am still not sure what to do even though reading what you suggested. Can you please tell me what to do following running the split.library script (according to nr 1 in my previous post, that is without removing the reverse primer)? If I denoise using n3phele, what should I do then in order to remove the reverse primer and adaptor?

Best,

Hedvig

[Tony Walters]

Hello Hedvig,

To do denoising and remove the reverse primer, do this:

1.  Run split_libraries.py (don't worry about the reverse primer removal at this step).

2.  Use output of split_libraries.py with denoising.

3.  Remove the reverse primers from the output of denoising with truncate_reverse_primer.py (http://qiime.org/scripts/truncate_reverse_primer.html).

The resulting sequences should be ready for the rest of the pipeline (starting with OTU picking).

Hope this helps,

Tony Walters

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[Hedvig]

Great - thanks a lot! I will do this!

Best,

Hedvig

[Hedvig]

Hi again,

I forgot to ask. Do I have to run the inflate script following denoising but before the truncate_reverse_primer_py?

Best,

Hedvig

Den onsdagen den 29:e augusti 2012 kl. 16:00:10 UTC+2 skrev TonyWalters:

[Hedvig]

Also,

I have run 2 lanes and I was thinking of doing what you suggested and then concatenate the two lanes? Should I do that following running the truncate_reverse_primer.py?

Best,

Hedvig

 

Den onsdagen den 29:e augusti 2012 kl. 16:00:10 UTC+2 skrev TonyWalters:

[Tony Walters]

Hello Hedvig,

For question 1, yes do the inflate step before the truncate_reverse_primer.py step.

If you are going to concatenate two lanes, that's fine to do with a cat <fasta file path 1> < fasta filepath 2> > combined_seqs.fna approach, I would suggest adding the -n command to split_libraries.py

so -n 1000000 for the first call, and -n 2000000 for the second, just to get unique enumeration for the fasta sequences when you combine them.

-Tony

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[Hedvig]

Ok, thanks I will do that!

// Hedvig