I was hoping to use Trinity, but it does not allow kmer size more than 32. Based on few expert suggestions, the ideal kmer size here should be between 75-100. Will Trinity based assembly be of suboptimal quality?
RNAseq isn't going to be as good as a genomic sequencing to get your assembly no matter what you use. You're going to get ambiguity which will mess with contiging because of the repetitive reads and the inconsistent read depth.
A larger k-mer helps, but is not necessarily better, there is such a thing as too high a kmer when assembling. Finding the sweet spot usually takes a few rough draft assemblies to dial in.
Trinity has served me well when I've not had the option of genomic reads, though. I would stick with it if you can't find a reference.
Hi Snehal,
If you have a reference genome of the organism then you can first align the RNA-Seq reads to the reference genome using STAR or BowTie and then do genome-guided Trinity assembly. This had worked well for my purpose.
All the best,
Swapnil.
Dear Swapnil,
Thank you for your answer. I will try this next.
Do you think the Genome-guided assembly will work in case the genome is of a related species but not of the same species? Please let me know if you had any such experience and whether it worked well in the end.
Regards,
Snehal
I am not sure. The quality of assembly with other related species might depend on the percent similarity between the two species and missing genes. You might have to play with mismatch cutoffs in STAR.