sequencing to identify novel restriction sites
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2.8 years ago

I am looking for a sequencing protocol and associates tools to discover one or more consensus sites from DNA fragments digested by a novel enzyme.

The template is plasmid sized and fully characterised, so I can map reads against it.

The fragments range from 10's of bases to 100's and are quite many so I will need to rely on sequencing depth to score the discovered motifs. The small fragments a

Does anyone have a primer or links? Thanks

sequencing • 488 views
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not sure I completely follow.

Do you have sequence data already or are you looking for a clever sequencing strategy to ease the post-processing?

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I am looking for a sequencing method to identify the restriction sites from processed template and derive consensus sequence(s) specific for that nuclease.

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Please use ADD COMMENT or ADD REPLY to answer to previous reactions, as such this thread remains logically structured and easy to follow. I have now moved your reaction but as you can see it's not optimal. Adding an answer should only be used for providing a solution to the question asked.

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Seems like RAD-seq to me?

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Corret. http://catchenlab.life.illinois.edu/stacks/

Not sure about this part though

and derive consensus sequence(s) specific for that nuclease.

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It is close indeed but RAD seq implies (as far as I understand it) further fragmentations and in our case we already have a majority of fragments smaller than 100bps. Therefore I think we will need to tag the ends somehow and make concatemers to be amendable to sequencing which otherwise would discard the small fragments with primers exclusion steps.

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You could recover the small fragments by acrylamide gels (pippin prep?), end-repair and then sequence?

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why not use a protocol suited for smaller fragments? and then do 50bp SE sequencing?

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same comment as above but you are right, short reads should be good enough. Thanks lieven ;-)

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