I am planning to use Oxford Nanopore's Flongle flow cell (via MinION) for targeted sequencing of 30 genes (with total length of 1.5GB). For this, I will first amplify all target regions, and the rough estimate is that around 100 primers (forward and reverse combined) will be needed to cover these genes.
What are the practical limitations or considerations when using 100 primers for multiplex PCR?
Is there any better approach to get targeted region of 30 genes than amplification by primers?
100 sounds like too much for a single reaction. A similar topic has already been discussed on RG, btw: https://www.researchgate.net/post/Amount_of_primersets_suitable_for_multiplex_PCR
However, unless your goal is to create a single-reaction assay for repeated use, why don't you just run several reactions with simpler mixes, and only mix the amplicons later in the procedure?
Also, I have seen hybridization (microarray) based methods used for much bigger gene sets, such as https://pubmed.ncbi.nlm.nih.gov/17934468/ or https://pubmed.ncbi.nlm.nih.gov/17934467/
I agree with Matej that it's better to run multiple PCR reactions with different primer sets. In the post they shared, the discussion assumes the primers won't bind to each other, but your 100 primers may well have dimerization that could inhibit the reactions.
Another consideration is that if you run a single reaction, depending on your product sizes, it may be difficult to know if all of your desired amplicons are present. It would be a bummer to spend the time and money on sequencing, only to discover that your amplification was only a partial success. By splitting out your PCRs into multiple tubes, you can ensure all your needed amplicons are successful before committing to sequencing.
You cannot use 100 primers in one reaction and expect anything quality or useful from that reaction. The time spent trying to optimize the reaction would be literal years. Talk with your advisor, what you are considering trying will not work and will cost a lot of $$$.
You could use 96-well plates and a liquid handling robot to set up 96 different pairs of primers in a single plate for each template. Put unique barcodes on each set of primers and you can use Illumina or other next gen sequencing. Or just bang out the sequencing using Sanger. Add M13 or T7 some other "universal" primer to all of your forward (or reverse) primers so you can use that universal primer for the Sanger. If you have less than 100 or so samples, I'd just use Sanger unless you *already* know how to do library preps and the data analysis.
BTW, I only noticed it now - covering 1.5Gbp length of 30 genes via ~50 PCR products is not possible. PCR is extremely inefficient for products more than a few thousand bp long! You can realistically cover only some fragments of these genes.
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