I have a number of miRNA precursors. These are the ones that mapped to a concatenated genome consisting of B. napus EST, B. oleracea EST and B.rapa genome. Some of the precursors have mapped to negative strand while others to positive strand. I was going through a paper saying that those precursors that map to positive strand of EST are taken as positive results.
Does the coding nature of positive strand has to play a role in this kind of selection? Although, I read somewhere that +/- sense of ESTs has nothing to do with transcription orientation rather on mapping of EST on genome!
It would really appreciate if anyone could please tell me their opinion on this.
Thanks.
Basically, people assumed ESTs as expressed transcripts, which means those sequences should be positive. When miRNAs are produced, they should mapped positively to expressed transcripts.
However, the assumption of EST must be positive is not necessarily true. EST library is made by sequencing end region of cDNA clone. The orientation of cDNA insertion in clone is not determined. So ESTs can be the same as expressed transcript (+) or reverse complement to expression transcript (-).
http://www.ch.embnet.org/CoursEMBnet/Zurich04/slides/EST.pdf
This slide can help you understand how ESTs are sequenced.
Also, when EST assembly (TGI databases http://compbio.dfci.harvard.edu/tgi/) is built, both positive and negative ESTs are used to build contig. It has nothing to do with transcription orientation.
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