Question: How to map RNASeq data to reference genome to check T-DNA insertions?
0
gravatar for Wuschel
5 weeks ago by
Wuschel260
HUJI
Wuschel260 wrote:

I used "Kallisto" to map RNASeq data to reference transcriptome and have done my analysis.

How I have a confusing outcome. Out of my 5 T-DNA knockout genotypes, two of them are showing the expressing of the gene (even higher than the WT).

These are published lines and I have already genotyped them by PCR before.

Is this because of the pseudoaligning?

How can I check this? Is there any way of mapping?

P.S. I do not believe there is cross-contamination!

I am not a bioinformatician and would appreciate it if there is a step by step detailed protocol to do this.

ADD COMMENTlink modified 5 weeks ago • written 5 weeks ago by Wuschel260
1

Try an aligner such as STAR and then check your alignment for T-DNA on IGV.

ADD REPLYlink written 5 weeks ago by Nicolas Rosewick8.3k

Thank you Nicolas,

Even if I see this T-DNA is there by this method, can I use this Kallisto o/p data for downstream analysis?

Since Kallisto is not giving reliable information on my mutant genes? I'm confused now...

ADD REPLYlink written 5 weeks ago by Wuschel260
1

could you post the output of kallisto for T-DNA plase ? and also could you specify which reference transcriptome you used ?

ADD REPLYlink written 5 weeks ago by Nicolas Rosewick8.3k

This is the reference: AtRTD2_19April2016.gtf. What do you mean by kallisto o/p? Which o/p ?

ADD REPLYlink written 5 weeks ago by Wuschel260

The abundance estimation for T-DNA (from the abundances.tsv file from kallisto quant)

ADD REPLYlink written 5 weeks ago by Nicolas Rosewick8.3k
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