Question: Structural variation detection from RNA-seq
0
gravatar for Hanan
19 months ago by
Hanan70
Tel Aviv University
Hanan70 wrote:

Hello I have two sets of reads that were obtained from RNA bulks of resistant and susceptible wheat recombinant inbred lines. I am looking for polymorphism between the bulks. Based on previous results, I suspect that the polymorphism associated with the resistance is a large insert of 1-5kb (up to the whole gene). I also suspect that this insert is not present in the reference genome. I am looking for a software that can detect large structural variations using de-novo assembly.
Can you please recommend which software to use? Thank you

rna-seq indel • 983 views
ADD COMMENTlink modified 18 months ago by colindaven1.2k • written 19 months ago by Hanan70

I don't think RNA-seq is the most appropriate technology for this, but now that the data is already generated we better make the best out of this.

I'll assume you don't have a reference genome since you want to go for de novo assembly?

Based on previous results, I suspect that the polymorphism associated with the resistance is a large insert of 1-5kb (up to the whole gene).

So you expect an insertion of an entire new gene? That gene should be expressed before you will see anything in the transcriptome, right?

ADD REPLYlink written 18 months ago by WouterDeCoster38k

Thank you for the reply. I expect an insertion of an entire new gene, I know that resistance genes have in many cases presence/absence variation. I expect that the gene will be expressed because the resistance was active at the time that the samples were taken. However, the expression levels may be low. I do have a refernce genome and I have conducted SNP and short indel discovery with this data. This got us closer to the target but looking at the refernce genome we found no candidate genes.

ADD REPLYlink written 18 months ago by Hanan70

An entire gene wouldn't map to the reference genome, I would try a de novo assembly on the unmapped reads.

Note that I haven't done anything like this before - it's my first intuition ;)

ADD REPLYlink written 18 months ago by WouterDeCoster38k

Yes, this is what I am looking for, de-novo assembly and comparison of long sequences.

ADD REPLYlink written 18 months ago by Hanan70

For RNA-seq the most commonly used de novo assembler is trinity

ADD REPLYlink written 18 months ago by WouterDeCoster38k

what do you mean with "looking at the reference genome we found no candidate genes"? did you see a large insertion or not? and why do you hope to see more relevant information from the transcriptome data?

ADD REPLYlink written 18 months ago by Friederike3.8k
1
gravatar for colindaven
18 months ago by
colindaven1.2k
Hannover Medical School
colindaven1.2k wrote:

Interesting project. I would approach it like this:

  • Trimming - very important pre de novo assembly
  • de novo assembly eg with trinity or Soap denovo trans or CLC or Abyss
  • extract ORFs with transdecoder
  • functional analysis of transcripts with interpro scan.
  • mapping to the genome with gmap
  • check mapped and unmapped and partially mapped genes in the area of interest - if known
  • orthology mapping - eg with proteinortho - for transcripts unique to one set. Join this information with the IPS scan info
ADD COMMENTlink written 18 months ago by colindaven1.2k

Thank you for the advise, I will give it a try.

ADD REPLYlink written 18 months ago by Hanan70
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