transcriptome with and without reference genome

Question

[backmoons]

Dear all,

I am new to bioinformatics and have a question for my project. I have three species that have close relationships, one species has a reference genome (I used hisat2 for this), other two species haven't had a reference genome, each species have about 7 time-point. I assembly the two species without reference genome using de-novo trinity method. But I just confused about what I need to do the next step? do I need to make a gtf file using transcriptome and mapped reads to the transcriptome, just like what I did for the species that have a genome? My goal is to find the differential expression genes and find the key-genes that related to our project.

I would be really appreciated if you could give me any suggestions.

Thanks

Answer 1

[WouterDeCoster]

To do a realistic differential expression analysis you need to treat the 3 species the same way, which is either map all to a reference genome or assemble all de novo.

Answer 2

[Kristoffer Vitting-Seerup]

I would suggest a two stage approach:

Step 1: Analyse each species (aka timecourse) separately using the best annotation you have. This will result in list of genes changing their expression over time.

Step2: Compare the differentially expressed genes using homology (compared to the reference transcriptome) to figure out which genes behave similar and which behaves differently.

Cheers Kristoffer

[backmoons]

Hi, I think this makes sense! But I am a little confused with step2, after finding the differential expressed genes within each species, we need to do the compared the homology genes? could you please give me more details about the step2..Appreciate it!

[Kristoffer Vitting-Seerup]

You would simply have to compare the sequence of the identified genes in one species to the sequence of the identified genes in the other species to identify which are so similar they can be called the same. With regards to specific tools for doing it I have to say I do not know - but asking a new question in the assembly section fo Biostars might give you something.