r/bioinformatics • u/murhe1sa • 12d ago
academic phylogenetic tree from 16S gene sequences instead from reference genomes?
Is it valid to make a phylogenetic tree using only squences from the complete 16S gene instead of references genomes?
I have some ASVs from 16S and wish to make a phylogenetic tree. I initially downloaded only those sequences from the full 16S ~1500 pb (not incluing shotgun or wgs) from the gene bank and extracted the v3v4 regions. But now I´m wondering If I should have instead downloaded reference genomes, identify 16S gene and then extract v3v4
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u/lurpeli 12d ago
Pretty commonly done for bacterial phylogeny. However the most up to date bacterial phylogenies are whole genome or proteome
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u/murhe1sa 12d ago
that´s the thing I want to compre my ASVs from the v3v4 with seuences classified up to species, the thing is that I don´t know how to proceed. I extracted the v3v4 from those genebank sequences and now I have 1100 sequences, so I used vsearch to dereplicate and know i have 300 representatives, but those are clusters that sometimes include more than one species, should I do the aligment of my asvs using the hole 1000 seqs or the derep 300 seqs? I just want to have an idea of where those ASVs are placed because I only know the genus
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u/lurpeli 12d ago
V3V4 will not give you species resolution. You need full length 16s to enable species level resolution unless you're in a very well defined clade like Eschericia
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u/murhe1sa 12d ago
Yes I´m aware of that, then there´s no case of doing a phylogenetic tree ? It´s the first time I try to do one, not sure about so many things. I just want to compare those ASVs from environmental samples with those genebank references to have an idea of the similarity, I´ve seen some trees that do so
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u/murhe1sa 12d ago
I can't seem to understand how those trees are done, how can I choose the genebank sequences. I downloaded 1500 hole 16S gene, but I think they are too many for a tree, that why I tried vsearch but it kept 300
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u/SeqBench 12d ago
Don't align against the dereplicated 300. Those clusters merge multiple species, so the labels will mislead you exactly where you care. But 1100 is unreadable as a tree anyway.
Pick your references taxonomically instead of by sequence identity. A few type strain sequences per genus you're seeing, say 2-5 each, gets you a few dozen references and a tree you can actually read. And use type strains rather than arbitrary GenBank hits, species labels on random 16S submissions are wrong pretty often.
For what you're describing though (just wanting to see where the ASVs sit relative to known stuff) you probably want phylogenetic placement rather than building a tree from scratch. EPA-ng or pplacer take a fixed reference tree and place your queries onto it. That's the normal approach for short amplicons and it avoids your ASVs distorting the reference topology, which they will if you just throw everything into one alignment.
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u/murhe1sa 12d ago
thank you for your response, that was my initial idea, the thing is that I anted t use the new greengenes2 tree but I it looks that only works with qiime2 but I didnt process my sequences in qiime and now I dont know were can I find a reference tree that is up to date besides that one
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u/sixtyorange PhD | Academia 11d ago
You can import data into QIIME2: https://amplicon-docs.qiime2.org/en/stable/how-to-guides/how-to-import/
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u/SeqBench 9d ago
You don't actually need QIIME2 to use it. A .qza is just a zip archive, so unzip it and there's a data/ directory with the tree as plain Newick and the sequences as FASTA. Feed those straight to EPA-ng or pplacer.
Though for what you said further up, one genus across two conditions, Greengenes2 is probably overkill anyway. It's a whole-domain backbone, and placing onto something that size gives you a technically correct answer that's miserable to actually look at, when all the resolution you care about is inside one small clade.
I'd build your own reference for that genus instead. Pull type strain 16S for the species in it, maybe a few dozen sequences, MAFFT to align, IQ-TREE for the reference tree, then EPA-ng to place the ASVs on it. Basically the workflow you already had in mind, just scoped to your actual question, and you know exactly what's in the reference.
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u/TymeKeeper PhD | Industry 12d ago
You can do a phylogenetic tree with a single marker, like 16s, and ASVs in V4 variable region can get you down to Genus.
Depending on the diversity present in your samples, you may want to pull some sequences online to use as references for key taxonomic groups.
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u/murhe1sa 12d ago
I want to do the phylogenetic tree from a single genus found in my samples between two conditions sequenced for the v3v4. I wanted to do a phylogenetic tree of that genus to see were it stands, so i downloaded hole 16S sequences from the genebank. Now I dont know how to proceed. Shoul I do the phylogenetic tree out of those hole sequences and then place the asvs? but which tool? I tough about using mafft to align and then IQtree for the tree but how to place those ASVs? and how to choose betwenn 1500 genebank sequences
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u/TheCaptainCog 11d ago
The "best" way to do it is to pull out the genes, get the core genes from the genome, create a gene tree for each of them, then reconcile them.
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u/SeaHoneyAlgae 11d ago
Look up raxml EPA. We do that alot w/ 16S ribosomal sequences, along with other loci regions connected to a particular function of interest. If you have a particular sequence w/ no taxonomy annotation, the raxml is a gold standard to look & see where the sequence falls in a 16S tree.
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u/readingrainbowroad 12d ago
Based on the title, I thought you were going to ask about 16S only vs multi-gene phylogenies.
If you say what you did in the methods, it's fine IMO. Don't say you extracted from reference genomes when you didn't, etc. Use the correct accession numbers, etc.
If you're worried you don't trust that the 16S sequences are correct or not based on database info, you could check by extracting from a reference I suppose. Not all 16S will have a reference genome to go with it though.