I have a general question about building phylogenetic trees without alignment:
I want to build a tree using an alignment-free approach by counting kmers. I have found a lot of information in the literature about this type of practice but I'm having trouble finding approaches that use Illumina short read sequences (which is what I have).
In my first attempt, I was able to generate a dendrogram by counting kmers and clustering them, but it did not group the sequences I had in the “correct/expected” way (these are known strains that are part of specific subclasses related to S. cerevisiae - in theory, this kmer clustering approach should’ve been able to group the strains based on their subclass pretty easily).
There are 21 samples in total with 5 specific subclasses. Does anyone know a way to either cluster or simply calculate the distance between kmers to generate a tree of 20+ Illumina short-read samples?
EDIT: The main reason for doing this in this way is to eliminate any bias from the reference sequence and maintain all the alleles (most of the samples are tetraploid) so I really want to avoid collapsing the data with any type of preprocessing alignment or assembly.
With that being said I would want to use the whole-genome short-read sequences of these 21 samples to count the kmers across the genome for phylogenetic analysis. If it gets too complicated, I would be okay with assembling the reads first before kmer counting.
I used Kitsune to determine the optimal kmer size for the samples which comes out to 15mer. I was able to count 15mers with all the samples using three methods: pure raw reads, assembling the reads with SPAdes, and downsampling the raw reads to 5x coverage. Using Jellyfish and custom scripts I wrote, which didn't end up being too taxing on my computer/server.
Currently, I have 21 '.tsv' files that list the kmer and the kmer frequency for each individual sample and I have one giant matrix that combines that data together so I have the kmer and the kmer count for each sample in one file:
I have looked briefly at kWIP. Ideally, I would use their distance calculation function to generate a .dist file for my giant matrix - I'll look into what inputs can be used for that function.