Hello everyone!
I have the following situation/design that I could use some help figuring out the best way to analyze:
I have two closely related species (non-model, no reference genome) that differ in their response to injury, and I want to compare gene expression along a 4-point time series and across both species. The basic experiment is to sample total RNA for RNAseq at four timepoints at each of the two species: a time t0 (baseline) and 3 consecutive time points t1 to t3.
My conceptual pipeline so far is as follows:
-Sequence total RNA using Illumina HiSeq 100PE
-Pool and clean up reads for time points t0 to t3 for each species.
-Assemble a transcriptome for each species using Trinity.
Now comes the question. I can analyze differential gene expression for each species along timepoints using RSEM/edgeR. But how would it be best to compare the same timepoints (say, t0 or t2) across species? It seems to me like I should somehow assemble a "consensus transcriptome". Any ideas on how to do it? My thoughts so far are doing a reciprocal blast search of both transcriptomes and use it to build a "join table", then use it to compare the results of the RSEM analyses made against the species-specific transcriptome; however, I am not sure if FPKM values obtained against different references are comparable.
I appreciate any thoughts or ideas on this!
Cheers!
-Ed-
I have the following situation/design that I could use some help figuring out the best way to analyze:
I have two closely related species (non-model, no reference genome) that differ in their response to injury, and I want to compare gene expression along a 4-point time series and across both species. The basic experiment is to sample total RNA for RNAseq at four timepoints at each of the two species: a time t0 (baseline) and 3 consecutive time points t1 to t3.
My conceptual pipeline so far is as follows:
-Sequence total RNA using Illumina HiSeq 100PE
-Pool and clean up reads for time points t0 to t3 for each species.
-Assemble a transcriptome for each species using Trinity.
Now comes the question. I can analyze differential gene expression for each species along timepoints using RSEM/edgeR. But how would it be best to compare the same timepoints (say, t0 or t2) across species? It seems to me like I should somehow assemble a "consensus transcriptome". Any ideas on how to do it? My thoughts so far are doing a reciprocal blast search of both transcriptomes and use it to build a "join table", then use it to compare the results of the RSEM analyses made against the species-specific transcriptome; however, I am not sure if FPKM values obtained against different references are comparable.
I appreciate any thoughts or ideas on this!
Cheers!
-Ed-
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