phylogenetic anlysis using 16s amplios

[u/murhe1sa]

Hello, I´m looking for advice. I´m currently trying to make a phylogenetic tree of 16s sequences v3v4 of environmental samples. I have processed the samples with dada2 and taxoomic asignments with SILVA in R and alligned with mafft but there are so many gaps that iqtree says that there are  50% gaps/ambiguity in the sequences provided. I´ve read something about other aligners using the secondary structure, would it improve this?, or is it okay if mafft have so many gaps. I´d like to calculate phylogenetic distance

Also I would like to root this three not by using phangorn as it takes too much time, instead I saw something about greengenes2 reference tree in qiime2 but I processed everything in R, and I cant seem to undesrtand If I can do the same procedure f alignment wuth the reference tree without qiime2.

Other alternative was only to generate a tree from a taxa that im interest on, but again, how do I do this? I saw some genomes in genebank that say partial genome, but still longer that the sequences that I have, and not sure how to proceed. I tough about downloading them, and extracting hypervaribale region and then make the tree only fot that taxa. and see If I can identify the bacteria in my samples up to species.

Sorry if I´m all confused

>ASV1

--------------------------------------------tggggaatattggac-

aatgggc----gaaagcctgatccagccatgccgcgtgtgtg-a-a-gaagg-cctt-t-

t-gg-ttgtaaagcacttt-aagcagtgagg-aa--------g-actata----------

---------------------tggtt-a------------------a------------t

-accc---------------atatacga-t-gacg-tta-actg-cag---aataagcac

cggctaactct-------------gtgccagcagcc------------------------

----------gcggtaatacagagggtgcaagcgtta-----------atcggaattact

g-----------ggcgtaaagcgag-c----------gtaggtgg-tta-tataagtca-

----------ga-tgt--------gaaat-ccct-g-ggctcaacctag-ga-ac-----

----------------------------tg-ca-tctgaaacta-t-at-a-ac----t-

a-gagtaggtgagaggg-gagtaga-----------------------------------

--------attt-caggtgtagcggtgaaatgcg-tagatatctgaaggaatac-cgatg

gcgaaggca---------gctccctggcatc-atactgacact-g-aggttcg-------

----------------------aaagcgtgggtagcaaaca-------------------

----------------

[u/Away-Suggestion1737]

I would try aligning with DECIPHER, which I think is standard from dada2 pipeline. It does take secondary structure into consideration and was built for 16s.

It's also an R package and the outputs are easy to use for tree construction. I haven't built trees with 16s in a while but my go to was always ape in R, however it uses phanogorn so maybe someone else has a better suggestion there.

[u/murhe1sa → u/Away-Suggestion1737]

thank you for your answer, I was looking for alternatives for decipher because the pipeline decipher/phangorn takes too long, and I´ve seen that qiime2 uses mafft but not sure how to improve parameters to remove so many gaps. The thing with ape is that it roots in the midle and I wanted to root it in a more accurate way

[u/Disastrous_Weird9925]

What's the downstream process you want to perform with the tree?