# How to get the longest fasta sequence including all possible switching isoforms of a gene out of isoforms

1. I am working with RNA-seq data without a reference genome/transcriptome and am instead using a Trinity de novo transcriptome assembly.
2. I analyzed both isoform and gene expression abundances using RSEM.
3. And got differentially expressed GENES via DESeq2. In the next step I want to get fasta sequences of differentially expressed genes to use them in the subsequent BlastX/Blast2Go analyses.

Considering the fact that my Trinity created assembly is based on isoform id:

How can I extract the gene sequence out of it? Is that a right thing to combine all the isoforms of a single gene in order to generate the longest possible isoform assembly?

If so, how can I do it to have a union-exon (combined form of all the exons present in the isoforms of an individual gene) sequence used in my Blast analysis?

If not, how should I proceed with the data type available? Trinity assembled file includes the isoform column as:

TRINITY_DN1780_c0_g1_i2
TRINITY_DN1780_c0_g1_i1
TRINITY_DN1780_c0_g1_i3
TRINITY_DN1780_c0_g1_i4
TRINITY_DN1780_c0_g1_i5


and the DESeq2 produces the output (gene differentials) as:

TRINITY_DN1780_c1_g1


From your question it isn't clear if you are trying to identify the isoforms that have a very high or very low differential expression value, or if you are trying to correctly find an orthlog for your top hits using BLAST.

If your question is about isoform-level quantification, identifying DE at the isoform level is challenging and DESeq2 is only designed for gene-level analyses. That is why it is only outputting genes rather than a specific isoform.

• Many thanks for your quick reply. I attempt to identify all possible differentially expressed transcripts (resulted from comparing the samples of tw) by Blast or Blast2Go approaches (since I have no reference genome/transcriptome in hand). If at gene level, my first problem is that I can't get the fasta files of gene level (DESeq2 output) from Trinity fasta file. Is there any automated software/command to get gene level sequence from Trinity fasta file? 2. If not, my second problem is that which procedure should I follow to analyze my data at isoform level (preferably for those with high diffe Dec 5 '18 at 14:46
• So, just to be clear - your final goal is to find the specific isoforms with the highest differential expression from your transcriptome? Dec 5 '18 at 14:51

There's no clear way to go from a trinity "gene" (this is just a cluster of isoforms with shared sequence) to a linear gene sequence that will be blastable and not cause a number of headaches (e.g., due to swapping the order of exons).

What I would suggest that you do is either Blast2GO the isoforms within the Trinity-defined "genes" or take the highest expressed isoform within a "gene" as the sequence you feed into Blast2GO for that gene. Alternatively just use isoforms for everything and avoid this whole issue.

There are a lot of questions in this post, and the main ones have been answered. Here is an answer to the one that has not been answered yet:

How can I do it to have a union-exon (combined form of all the exons present in the isoforms of an individual gene) sequence used in my Blast analysis?

I don't think you should do this, but if you want one of the usually many plausible linearized representations of transcript isoforms, then Lace would do the trick.

It appears from the comment in another question that this question is also about transcript-level differential expression analysis.

If instead of using RSEM you use kallisto for transcript quantification, then you could use sleuth to perform transcript-level differential expression analysis.