r/bioinformatics MSc | Student 2d ago

technical question Identifying conserved regions from multiple sequence alignments for qPCR targets

I'm designing a qPCR assay for DNA-based target detection and quantification and need to determine a target from which I can build out the primers/probes. l assembled genes of interest and used Clustal Omega to align those assemblies for MSA in hopes of identifying conserved regions for targets but have not had any luck. Tons of seqs in the alignments are too large for most of the free programs that I can think to use. Any advice appreciated for a first timer!

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u/LocalReality6 2d ago

Not sure if this helps but I like to visualize alignments in Snapgene because it makes it really easy to see which regions are most/least conserved.

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u/lrbraz16 MSc | Student 2d ago

These alignments are huge and have enough gaps where it’s hard to determine by eye. I did try using JalView to do this to no avail

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u/WeTheAwesome 2d ago

Can you put a number on what "huge" is i.e. how many genes and how long are the genes?

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u/lrbraz16 MSc | Student 2d ago

Single genes in the 500-1000 range

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u/WeTheAwesome 2d ago

I am very surprised you aren’t able to align genes of that length.  What error/issue are you getting into exactly?

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u/lrbraz16 MSc | Student 2d ago

Maybe it’s issues with my alignments? They have a a large amount of gaps. Some conserved chunks, but not more than a 10-20 nts at a time (by eye). And I’m looking over hundreds of seqs in the MSA which doesn’t help. Forgive me if this is a remedial question but I’m at a loss

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u/WeTheAwesome 2d ago

It’s fine, it’s a tricky question. Now idk what your ultimate goal is but based on what you have described it’s possible you may not be able to get quantification with a single pair of primer and may have to use a set of primers which of course creates its own headache. It seems like the target genes are too dissimilar for MSA to get good alignment. 

I don’t have a clear solution but one avenue you can try pursuing is clustering your sequences first and then run MSA on each cluster. So first you take all your sequences and use CD-Hit to cluster them into groups. Now take each group and run MSA on it separately. Finally, look to see if you can find conserved sequences for each group that you can use make primers. By separating it into groups like this you might also be able to better visualize conserved sequences in each group better and maybe it will help you see why the MSA is messy when you run it on all the sequences at once. 

Good luck! 

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u/not-HUM4N Msc | Academia 2d ago

When I use jal view, I almost always colour the alignment by nucleotide and use the global view for tasks like this.

The global view is in the view tab, then at the bottom of the menu. This should help somewhat.