Question: RNA-seq: differentially expressed genes without genome reference
2
gravatar for guiu
3.5 years ago by
guiu60
Luxembourg
guiu60 wrote:

Hello,

I have RNA-seq data for an organism for which the transcriptome is available, but not the genome (it is not assembled yet). I am interested in differential expression analysis at gene level.

I have been reading about RSEM, CuffDiff, Sailfish, Kallisto, HTSeq, but I didn't get a clear idea of which is the best approach to get differentially expressed genes.

What is your recommendation? Thanks

 

rna-seq • 2.0k views
ADD COMMENTlink modified 3.5 years ago by Rob3.4k • written 3.5 years ago by guiu60
6
gravatar for Rob
3.5 years ago by
Rob3.4k
United States
Rob3.4k wrote:

I'd recommend using Sailfish or Salmon with tx-import to get fast accurate gene-level results (example). tx-import will allow you to import these results directly into DESeq2 (or prepare them easily for most other downstream DE packages).

ADD COMMENTlink modified 11 months ago by RamRS23k • written 3.5 years ago by Rob3.4k
3
gravatar for Antonio R. Franco
3.5 years ago by
Spain. Universidad de Córdoba
Antonio R. Franco4.1k wrote:

You have two choices in the absence of a reference genome 1. Annotate you transcriptome with tools like Trinotate, do the mapping taking into account this information (Bowtie2, but, kallisto), and then the DE analysis (limma, DESeq2, edgeR, NOISeq) 2. Do the mapping and DE analysis and annotate afterwards

ADD COMMENTlink modified 3.0 years ago • written 3.5 years ago by Antonio R. Franco4.1k
1
gravatar for Kamil
3.5 years ago by
Kamil1.9k
Boston
Kamil1.9k wrote:

If you know which transcripts belong to a gene, then you can sum their TPM values to get the gene's TPM.

Consider using Kallisto to get quick and accurate results.

Next, use your preferred method for differential expression analysis: limma, DESeq, edgeR, etc.

ADD COMMENTlink modified 11 months ago by RamRS23k • written 3.5 years ago by Kamil1.9k

Do you I need annotation for that ? or only transcriptome is enough ?

ADD REPLYlink written 2.8 years ago by Picasa460

Kallisto requires sequencing reads and a file with target sequences.

Please see the kallisto manual for more information.

ADD REPLYlink modified 11 months ago by RamRS23k • written 2.8 years ago by Kamil1.9k
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