Advice on 16S Alignment for genus-specific qPCR design?
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2.0 years ago
Tbr • 0

I have been trying to develop qPCR assays for identifying certain genera of bacteria from environmental samples. I am decided to use 16S sequence data in order to find highly conserved regions of the 16S gene across the genus of interest, then compare it with closely related genera and families in order to identify potentially unique regions for my genus. I initially attempted looking for conserved signature genes and indels but found they were not generally appropriate for qPCR assay design due to the potential for false positive results.

However, I am not especially familiar with a lot of phylogenetic bioinformatics and I am not too sure where to start... Originally I tried retrieving the sequences from SILVA and aligning them however I just found that I got a little overwhelmed with the volume of sequencing data and I don't really know how to go about sorting the alignments into information that I can work with.

I am pretty new to the field of bioinformatics and am well aware I may be asking a pretty obvious or uninformed question, so any feedback or advice would be highly appreciated. Alternatively, if there are any alternate strategies I am happy to listen as well.

16S Phylogenetics qPCR • 445 views
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That's a tough one. I'm not sure the 16S is your best shot here, it might not have the resolution you're looking for, you'll probably have better luck identifying a conserved genus specific genomic region. A good place to start would be checkM taxon specific database: https://github.com/Ecogenomics/CheckM/wiki/Workflows#taxonomic-specific-workflow

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