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Old 07-10-2018, 08:12 AM   #1
seqtechno1
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Smile ddPCR for quantification of Illumina TruSeq library

Hello,

I am planning on using the BioRad Truseq ddPCR assay for Illumina quantification of a truseq cDNA library I made from RNA. The protocol calls for running dilutions of library in 10^-6, 10^-7, and 10^-8. The thing is, I am starting with very low concentration library to begin with, maybe on the order of 300pg/ul. Therefore, I am thinking of instead running something like 10^-2, 10^4, and 10^-6 dilutions instead, to make sure I don't over-dilute. Thoughts?
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Old 07-11-2018, 12:15 AM   #2
austinso
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Quote:
Originally Posted by seqtechno1 View Post
Hello,

I am planning on using the BioRad Truseq ddPCR assay for Illumina quantification of a truseq cDNA library I made from RNA. The protocol calls for running dilutions of library in 10^-6, 10^-7, and 10^-8. The thing is, I am starting with very low concentration library to begin with, maybe on the order of 300pg/ul. Therefore, I am thinking of instead running something like 10^-2, 10^4, and 10^-6 dilutions instead, to make sure I don't over-dilute. Thoughts?
Let's assume that your average fragment length is 300bp + 150 bp adaptor.

So, 300 pg/uL is 300e-12/(660*450)*6.022e23 ~ 6e8 molecules/uL

You want to be around 2e4 copies/uL in the ddPCR reaction, so the stock should be drawing from should have around 2e5 copies/uL.

So a 10^4 should be more than enough.

That being said, you may not have enough library depending on which sequencer you are using and how much volume you have of your library.

At least 10 copies/uL measured from a ddPCR reaction using a 10^6 dilution is about where you need to be.

Just be sure that the ddPCR assay is compatible (i.e. the right version) with the TruSeq version you are using.
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Old 07-11-2018, 07:04 AM   #3
seqtechno1
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hi austinso,

thanks for the information. i'm going to run through the calculations so that i understand the math better. i will have enough for sequencing because i can normalize all of my libraries to 0.5nM, then just add more volume of these to equilibrate the relative abundances. i've already done those calculations. the ddPCR was just a learning exercise to make sure our primers and probes and everything are working. we got some nice (looking) data out of it, but i haven't analyzed it yet. it looks like 10^-6 was the most informative dilution.
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