design primer for alternative splicing events

Question

[pengchy]

Hi,

For alternative splicing primer design, there have been several software available. But the needs are still not be satisfied. I want to find a stand alone software 1) with command line scripts that run on linux platform, 2) design event specific primers.

RASE [1] only designed for human and not provide stand alone software. RASE can design transcripts specific primers.

PrimerSeq [2] run on Windows and Mac, not command line available. And can not design the events specific primers. The primers span the events.

Reference:

[1] Brosseau, J. P., et al. (2010). "High-throughput quantification of splicing isoforms." RNA 16(2): 442-449.

[2] Tokheim, C., et al. (2014). "PrimerSeq: Design and visualization of RT-PCR primers for alternative splicing using RNA-seq data." Genomics Proteomics Bioinformatics 12(2): 105-109.

[John]

When it comes to designing primers that target alternatively-spliced transcripts, I would say a lot of the tools fall short of real-world applicability. Normally companies like PrimerDesign (http://primerdesign.co.uk) will manually design and test hundreds of thousands of primers so that when you buy the probes (something you have to do anyway) the primers are guaranteed to work.
I mean, by all means keep looking for some free software if it's out there - but personally i'd go with experimentally validated probes over silico probes if possible.

[mfahim]

Hi Pengchy.

Could you resolve the issue?.

I am facing a similar issue.. I need to design primers to differentiate various novel isoforms from the constitutive ones in Arabidopsis.

What would you recommend?.

[pengchy]

Hi mfahim,

I have resolve this problem with a perl script that specify specific primer region, especially can locate across the junction, and products length. Then send the request to primer3 package, which has many customized parameters for alternative splicing primers.

The input of my script are:

1. class event in the format of the output of gff_make_annotation.py PYTHON script from rnaseqlib package
2. the transcripts sequence

You can have a try and I am pleasure to share my script to you.

[John]

But have you tested the primers experimentally?