Adapt method for targetted tiled sequencing?

[matbeale]

Not so much of an issue as a query. I'm interested in using the tiled multiplex PCR approach for enriching targetted regions of larger genomes (e.g. bacterial) from mixed samples. I'm thinking about picking say 10-20 regions in different parts of the genome, each around 1Kb (so potentially wanting multiple amplicons per region). Can PrimalScheme cope with that type of approach, or can it only design for a contiguous sequence alignment? What would be the best way to input an alignment in this situation - a concatamer of different regions (which I expect would result in some bad designs) or can it take different (very divergent) sequences one after another, yet still part of the same overall scheme? Thanks

[joshquick]

Hi, We are working on that feature now and have it partially done. Should be able to give you an update in a week or so. Josh

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On Fri, Feb 26, 2021 at 7:15 PM ***@***.***> wrote: Not so much of an issue as a query. I'm interested in using the tiled multiplex PCR approach for enriching targetted regions of larger genomes (e.g. bacterial) from mixed samples. I'm thinking about picking say 10-20 regions in different parts of the genome, each around 1Kb (so potentially wanting multiple amplicons per region). Can PrimalScheme cope with that type of approach, or can it only design for a contiguous sequence alignment? What would be the best way to input an alignment in this situation - a concatamer of different regions (which I expect would result in some bad designs) or can it take different (very divergent) sequences one after another, yet still part of the same overall scheme? Thanks — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#26>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABC6R47M35ROQMWJYCJDO7TTA7XN7ANCNFSM4YI65QLQ> .

[matbeale]

Thanks Josh - I look forward to the update.
Best wishes,
Mat

[emdyess]

I have the exact same question! Any update on the implementation or whether there is a suitable workaround? If we can't supply a multi-fasta, could we concatenate with, say, 100nt spacers (e.g. N's)? I am looking to perform a single-reaction multiplex PCR.

[aresti]

We have this feature implemented and in a working state on this development branch:
feat-panels

If you clone and checkout that branch, then follow the 'install from source' instructions - you can try it out
Run as before except you can now pass multiple input files

It appears to be working fine and all the existing tests are passing. We need to tidy it up, write a few more tests and add some polish, before releasing it in the coming weeks. Web interface will follow shortly afterwards.

Consider it alpha for now but feel free to try it

[emdyess]

Wow! Thanks for the prompt response! I'll give it a whirl.

[aresti]

let us know how you get on - forgot to say, I don't think we got round to fixing the svg/pdf plot yet, so ignore that