I have a fairly simple objective. Our group has sequenced 2 unknown organisms and 1 known organism (all eukaryotes). We suspect they may be closely related but we're not sure. My idea to check this was to use Kraken2 with a custom eukaryotic database and perform k-mer classification on all the reads. I was hoping to get an output that would show me how similar the organisms were, but I came across this article that showed that kraken2 struggles at the species level as the refseq database continues to grow (https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1554-6). Because this is a lowest common ancestor program, I'm worried it will just output the same LCA at the genus level for all 3 organisms, in which this approach isn't suitable to figure out the similarity between all the organisms.
I'm wondering if anyone has any better suggestions for how to go about this study?
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Mark •
3 answers
Haven't tried it myself yet, but Read2Tree might be for you, read more at https://www.nature.com/articles/s41587-023-01753-4
I tried and really liked ASTRAL waster-site recently as it is easy and fast (assuming closely related taxa) - https://github.com/chaoszhang/ASTER
It was a lot quicker than read2tree and more relevant for my task at hand (intra species check).
I suggest assembling the mitochondrial genome or the nuclear rDNA operon with GetOrganelle (https://github.com/Kinggerm/GetOrganelle) and then conducting a phylogenetic analysis based on these sequences.
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You could compare the three organisms directly to see if they appear to be similar by using a kmer based tool like sourmash.
Such an analysis may give incorrect results if contamination is present.
In event of contamination any/all analyses are going to have problems.
GetOrganelle, which I recommended, will make the consensus sequence of the mitochondrial genome and nuclear rDNA. If the contamination is not extremely abundant, it should not pose a problem.