Hi,
I'm an undergraduate doing an internship so I'm sorry if I'm not explaining everything well or forget to add certain crucial information.
So for my project I had to clone 4 different constructs into plasmids and they all contained a fusion protein with a linker in between. After traditional restriction and ligation cloning, transformation and colony PCR we sequenced the promising clones (we use Eurofins Mix2Seq kit which is an overnight sequencing platform based on dideoxy chain termination). But in every single clone for all 4 plasmids, part of the linker is deleted according to the sequencing results (see image, top and bottom sequencing results show different plasmids). It is not always the same part for each clone from any of the plasmids, but multiple sequencing reaction with different primers on the same clone, does give the same deletion.
Something else I've noticed is that whatever part is deleted, the proteins always remain in frame.
For the transformation we used Stbl3 E.coli (which are supposed to have reduced recombination).
So I'm thinking it might be some sort of recombination issue or maybe a sequencing issue (I read that high-GC can be problematic, but the sequencing of the initial plasmid where we got the construct with fused proteins from, showed no problem in sequencing the whole linker).
Again sorry if the explanation is unclear, feel free to ask additional questions and thanks a lot in advance for any suggestions!
There is a lot of GGC motifs around the area of the sequence that is deleted. I think that a mechanism like G quadruplex where runs of G or GCs are annealing on the same strand to form a loop and then the sequencing enzyme reads across the neck of the loop to give an excised sequence. This secondary structure can be minimised either by sequencing at a higher temperature than recommended in the sequencing kit or by sequencing with dmso added to the sequencing reaction. Added betaine also works to clean up secondary structure but needs the salt removed prior to loading onto the capillary or you get very little and late starting sequence. If your sequencing kit contains a high GC buffer ( like many pcr kits do) then use that buffer. If Not then find an enzyme around the lab that has a high Gc buffer and add that to the sequencingreaction.
You may want to check that this is a sequencing artifact and not a pcr error with a suitable restriction digest of your sequencing template to check fragment sizes are as they shoud be. It would be interesting to see an original .abi sequence file to see if the correct sequence is a low intensity backgroung sequence under the strong signal indicating that some sequence is possible in the loop region
Our lab has the same issue many years ago (~2010), we are cloning the entire genome of arenavirus, it contains the IGR structure (lots of GCs). it took several months to make it correct by a very senior postDoc.
not sure whether seq error or a cloning error. you said all the seq results showed the same deletion, I think it might be a cloning error.
Paul Rutland, I've added a representable sequencing reaction (ab1 file). To me it seems good quality, also after the linker. I did try to sequence with 5% DMSO added, but this did not make any difference to the sequencing quality. Any of the other things you suggested I can not try since we use a service to do our sequencing. I personally don't think it's a sequencing issue because of the same reason as Junjie Shao said, for the same clones the part deleted in the linker is every time te same, even tough it sometimes differs between the clones.
In the end it's not a very big deal since the fusion proteins remain in frame, I was just wondering what the reason could be.
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