Entering edit mode
5.6 years ago
agata88
▴
870
Hi all!
I have 12 scaffolds that need to be connected with Sanger PCR products. I would like to design starters for all sequences. Is there a tool that can do that automatically?
I've tried CONTIGuator - it didn't help, cause my novel bacteria and it's related species reference sequence just don't match. The output is "No contigs mapped to reference! Exiting..."
So, is there a tool that can automatically design starters and fill gaps between scaffolds without genome reference?
Many thanks for any suggestions.
Agata
Primers?
yes, starters = primers
You just need a script which gets ~20nt from each end of the contig, and then reverse complement the 5’ one (since you’re doing an outward sequence). You don’t need a reference.
Please note that every contig ends in a repeat. If you place primers on repeats, you will most likely get all kinds of weird PCR products.
Maybe not every contig, it could just have been a low quality region for reasons other than repetition, but good point! You can design longer primers than 20nt which will help a bit potentially.
The real answer here is run it on a Nanopore machine and make a hybrid assembly.
Please, Correct me if am wrong. I have 12 scaffolds, for each I designed two primers at the beginning (reverse) and at the end (forward). I need them to go outside sequence. Now, I have 24 starters. Since I don't know what is the scaffold orientation, I need to pair everyone with everyone and run PCR. This is 552 PCR reactions. I am wondering, if I can run single primer PCR - and end up with only 24 PCR reactions?
Many thanks for any suggestion, Best, Agata
On which device do you intend to sequence? Perhaps you could use a multiplex PCR and just throw all primers in the same mix.
Sanger, nice point! I forgot about multiplex.
Well, Sanger makes multiplex PCR a lot harder, but you can still do the PCR in multiplex and then use each primer separately to do the sanger sequencing, I guess.
If it was illumina you could just amplify and sequence.