Question: sequencing to identify novel restriction sites
0
gravatar for Stephane Plaisance
5 weeks ago by
Leuven area (Belgium)
Stephane Plaisance380 wrote:

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 • 124 views
ADD COMMENTlink modified 5 weeks ago • written 5 weeks ago by Stephane Plaisance380

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?

ADD REPLYlink written 5 weeks ago by lieven.sterck3.9k

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

ADD REPLYlink written 5 weeks ago by Stephane Plaisance380

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.

ADD REPLYlink written 5 weeks ago by WouterDeCoster36k

Seems like RAD-seq to me?

ADD REPLYlink written 5 weeks ago by WouterDeCoster36k

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

Not sure about this part though

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

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by genomax62k

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.

ADD REPLYlink written 5 weeks ago by Stephane Plaisance380

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

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by genomax62k

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

ADD REPLYlink written 5 weeks ago by lieven.sterck3.9k

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

ADD REPLYlink written 5 weeks ago by Stephane Plaisance380
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