I have a large set of cyanobacterial gene sequences aligned and am trying to find potential primer sequences for them. I would like to create a multiplex qPCR with no more than 5 primer sets. The dataset has gaps and uncertainties. How do I go about grouping the gene sequences and designing primers to target the target regions in similar sequences? I used Clustal_X to align the sequences.
Dear Garrett,
What would be the aim of your multiplexing? Are going to detect ANY of the cyanobacteria species? or you will do specie identification, at least one of 5?
Besides, which gene sequences have you aligned??? are they of the same or related or distant species???
I would advise to reduce the number of sequences and leave only those which are reliable, associated to well species, as well as other characteristics would help you to improve the alignment. Besides, later you may use obtained multiple alignment in comparison to protein sequence, if the region corresponds, or intergenic regions, which are relatively small. This step, probably, will give you the hint how to group target regions.
So use biology knowledge to make sense of what you process. Thus, you can avoid situation of "garbage in" - "garbage out".
Good luck!!!
Levitchi,
Thank you. To expand, the goal is enumeration using the qPCR primers. I aligned sequences of a well-conserved gene, and the dataset contains both closely related species and more distantly related clusters. I'm hoping to use phylogenetic trees to help inform which groups I'll try to target with each primer pair. And I'll definitely have to remove some garbage sequences, haha.
That being said, could you elaborate upon comparing my multiple alignment to protein sequences to find target regions? Or if there's literature you could point me to that describes a method, that'd be great.
Thanks again.
Dear Garrett,
It would be nice to define your goal as otherwise you will need less specific primers and their elaboration is too tricky.
I dont think you need more literature.
You can use studying material from NCBI - https://www.ncbi.nlm.nih.gov/tools/primer-blast/.
What will be important is to clarify how primer designer tools works and which are the rules to accept/reject the results, as Primer3 (http://simgene.com/Primer3), Primer BLAST, or use the tools provided by different companies online, e.g. ThermoFisher, Eurofins (https://www.eurofinsgenomics.eu/en/ecom/tools/qpcr-assay-design/).
It will be important to know which will be your reagent provider, as then you can ask them for assistance in design.
Good luck!
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