Sequencing Through An Hairpin Structure
Hi,
we are having a problem sequencing a certain region of the genome. Apparently a hairpin loop is built, when the sequence is single-stranded, but it might also already be there under double-stranded conditions. So though we are doing a targeted sequencing of a specific region and have a coverage of over 5000fold, we still get a very low coverage of exactly that region, where we would like to find our results.
Does someone has any idea, as to how to solve this problem? Is there a way of preventing the building of a hairpin loop during the sequencing process, or of denaturing the sequences in the preparations?
Danke A.
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Neilfws
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Assa Yeroslaviz
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Assa Yeroslaviz
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wich sequencing technique did you use ?
This is a molecular biology question, not bioinformatics. Better to ask in an experimental molecular biology forum.
@NicoBxl - we used SOLiD
@neilfws - yes I know that, but bioinformatician are the guys who needs to work with the data, find out where the problems are and than try to find solutions for those cases. At least this is how I see it. The biologist comes to us also for this kind of results.
I don't think this is so off topic as you says.
Did you try to evaluate whether the sequences really are able to form hairpins? Did you look for restriction enzymes that could cut the sequence in such a way that hairpins would no longer form. That would be at least some ways in which bioinformaticians could help with this problem.
Yes we do know, that it can build hairpins. The question still needing answer is whether it is happens only in a single-stranded way or also while the DNA is in double-strand mode. About that there are different opinions. About the restriction enzymes, we also thought about it, but unfortunately there are long homo-polymeric stretches of 'A's in there, but no RE known to cut into them.