We frequently have mixed results for a qPCR assay. We'd like to determine if this is non-specific amplification, or target amplification when the target is low-frequency. DNA tape-station shows multiple bands, that are unique to different samples. Bands are too close together to reliably isolate. I'd like to sequence them all using next gen. I'm trying to figure out if this could work using the Ampliseq workflow for ion torrent, which is something we already do regularly. Distributer will not advise so I'm asking here. Thnx.
No, qPCR products cannot be sequenced in the Ampliseq workflow. The Ampliseq workflow is a targeted sequencing approach that uses hybridization-based capture to selectively enrich specific regions of interest in genomic DNA. On the other hand, qPCR (quantitative Polymerase Chain Reaction) is a widely used technique for quantifying specific DNA or RNA sequences, but it does not produce a sequencing-ready product. To sequence the qPCR products, they need to be amplified through PCR and purified before being used as the template in a sequencing reaction.
In this case, I would say T-RFLP could be a better or simpler choice rather than sequencing.
So just to understand you correctly, Abdulaziz, you believe the can be sequenced provided they are purified and cleaned prior to ligation? This was my plan. My understanding is that Ampliseq primers contain Uracil in the place of Thymine. I was thinking about ordering Ampliseq primers complementary to the qPCR primers, and performing a few rounds of PCR to incorporate them into the amplicons. From here the FuPa digestion (which digests the Uracil-containing primers), and then the normal cleaning and normalization steps that all DNA libraries undergo following amplification prior to Ion Torrent, and of course the ligation of barcodes and adaptors. This is what I meant by the Ampliseq workflow. Please advise, this should work, correct?
The distributor says it would not work but will not explain why, and instead wants me to purchase another kit for a new workflow. I am trying to understand if there really is some reason qPCR product cannot be sequenced (after library prep), or if they are just trying to sell me something.
RFLP won't give me the answers I am seeking. Past literature suggests genomic instability in WBC can result in multiple versions of the fusion gene our qPCR is intended to amplify. This isn't thoroughly explored in the literature, however. I will need to sequence to be sure, and would like to be able to classify what types of changes are being made that result in each patient having slightly different banding patterns on the tape station. These changes could be small indels or intron fragments that make it into the transcripts occasionally.
Cannot do sanger because we retrieve multiple bands that are too near each other to isolate. I'd like to have a look at all of them. We can perform NGS in our lab. We are a clinical lab and I am wondering why it should not be possible to put one sample of qPCR into our NGS workflow in one of our other assays just to get a quick look. After getting an idea of what is there (nonspecific binding or higher genomic instability of target than previously thought), we can make more detailed decisions about how to approach troubleshooting.
I want to use NGS to sequence the qPCR products from some of our patient samples for troubleshooting. These are cDNA, but DNA nonetheless. To me, this should be reasonable to prep as we would any other DNA amplicon for NGS sequencing. I've heard it cannot be done but no reasons as to why. I'm asking here for clarification on this matter.
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