denovo transcriptome assembly

Question

[1234anjalianjali1234]

hellow all,

I want to check the differences between two physiologically distinct roots in a plant (do not have reference genome). I have done RNA sequencing using novaseq, and want to do differential gene expression analysis, but i am confused in denovo transcriptome assembly. Do I need to have single assembly using both the samples or two different assemblies respectively and then do the mapping part to find DEG.

Thank you

[GenoMax]

two physiologically distinct roots in a plant

What does that exactly mean? Is it still the same plant species? Are they two samples from two parts of plant roots?

[1234anjalianjali1234]

Same plant species producing two physiologically distinct roots. Two samples from two different plants of same species.

[GenoMax]

Combining the two samples during de novo assembly should give you additional coverage/a more comprehensive representation.

You keep referring to both samples so is this an n=1 situation for both samples?

[1234anjalianjali1234]

two kind of samples with triplicates so its n=3.

Thankyou

Answer 1

[Kristoffer Vitting-Seerup]

The best approach is to generate a combined transcriptome from all your samples. You can read more about it, along with some suggestion for bioinformatic tools here.

Answer 2

[colindaven]

I haven't tried it, but this program might be helpful. https://genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1284-1

Generally, I'd use Trinity to assemble all transcripts de novo (combined), then try to map each replicate read set to the assembly.

Interproscan is quite good at assessing functionality using ORFs etc from Transdecoder.