Oligo Design From Ests
When designing oligos for a microarray from ESTs, it seems to be crucial to choose the correct direction (strand) for the oligos, but I can't seem to find anything in the literature on this, or how to do this. (I've written a small tool to conditionally reverse-complement ESTs using a dynamic programming algorithm that takes into account BlastX hits and poly-A etc, but I'm unsure how important this is for the results.)
Any pointers or opinions?
Correct strand is certainly crucial - wrong strand = no hybridization!
I guess there are 3 approaches:
if a researcher generates the ESTs in their lab, presumably they know whether they are 5' or 3' clones
if ESTs are obtained from
dbEST, sequences are annotated as 5' or 3' otherwise as you say, a software tool to match ESTs to chromosomal sequence is required
This looks useful:
"ESTPiper – a web-based analysis pipeline for expressed sequence tags". It includes a tool for microarray oligonucleotide probe design and the paper discusses other EST analysis tools, some of which also do probe design.
Google search for
EST strand probe design also throws up plenty of useful-looking results.
1) There is SeqClean utility which may help you get rid of artifacts:
You may also try to construct rRNA library and for using it with SeqClean.
2) check your ESTs for common repeats. While some transposons are expressed, I am not sure if you want to have some pre-mRNA intronic sequences on the chip.
3) did you tried assembling all your ESTs?
4) you may also tblastx your ESTs against these from related species. There may not be any proteins @NCBI covering the less conserved protein parts from your species of interest.
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