Im working on a metagenomics 16s rna project , a problem popped up that i have 20 sample each sample is one unique species. My aim is to extract 16s from while genome then identify the species and make phylogentic tree. I tried phyloflash tool to pick up th 1500bp but it faild to denfine most of them , then i used spades tool to assemble the genome and i checked the contamination using chekm2 10 of them has contamination less than 5% but the other 10 had 99% of contamination. Then i used metaxa2 to check where the contamination comes from and it was mainly from the mitochondria and the chloro plaste , so I tried to removing the contamination using mitochondria and the chloro plaste database and i used spades again to check the contamination nothing happened and it still the same. Also I found four samples with fragmented sequences after extraction, each is less than 600 bp so it's diffcult to identify the species from it or another fragment, I tried many tools to get longer sequence but I failed, also I found three of them with same genus and I couldn't get the species, so could anyone help me with this problem using 16s rna from whole genome?
1 answer
I think the premise is the problem. 16S is a bacterial and archaeal gene - plants don't have one in the nuclear genome. The 16S metaxa2 found in your chloroplast isn't contamination, it's the only 16S a plant sample has, because the organelles are of bacterial origin (metaxa2 will pick up the mitochondrial SSU alongside it). So you've been filtering out the thing you were looking for.
checkM2 doesn't apply either. It scores completeness and contamination for bacterial and archaeal genomes, so 99% contamination on a plant assembly isn't telling you anything real.
For plant species ID and phylogeny the usual markers are rbcL and matK from the chloroplast plus nuclear ITS or ITS2, and all of those are reachable from Illumina WGS. GetOrganelle will assemble the chloroplast genome from your reads and you can pull rbcL and matK out of the annotation; the nuclear rDNA cluster gives you ITS. ITS is also your best shot at the three samples stuck at genus level.
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what kind of genome sequence data do you have? illumina? ONT? ...
Which species are we talking about here? plant it seems?
Often people use dedicated approaches to do 16s analysis on metagenomic data, by which I mean they usually run a sample prep etc specific for rRNA (eg. amplicon sequencing of the rRNA regions, rather then WGS )
Whole genome sequencing from Illumina and they're species from plants, could you elaborate your answer as I've WGS not amplicon one?