So the dogma goes:
Use as few PCR cycles during library construction as possible.
One can imagine various rationales behind this one.
But is #3 even true? Before delving into it, I would like to exclude issues having to do with overrunning the supply of reactants in the PCR. If the amount of final product approaches the total supply of dNTPs in the reaction, I can easily imagine higher levels of misincorporation.
By "errors" here, we mean errors per total bases, right? If the PCR polymerase used is like Taq polymerase it likely has an error rate of about 1 in 10,000 bases polymerized.
Templates:
One thousand 100 base amplicons.
The question: will the error rate per amplicon be higher after 20 cycles than it was after 10 cycles?
Again, presuming reactants are not limiting and each cycle is 100% efficient -- that is, doubling the number of amplicons: After 10 cycles there will be 1 million (2^10 * 1000) amplicons (~0.1 pg of DNA). After 20 cycles there will be 1 billion (2^20 *1000) amplicons (~0.1 ng of DNA).
Doesn't intuition tell us that after 10 additional cycles the errors will have compounded and the overall error rate we might detect by sequencing 1000 of the amplicons will have increased?
Ignoring indels, I don't see this is the case. Sure, Taq polymerase would tend to misincorporate a base in 1% of the product strands that it creates, and that erroneous template will then be amplified each cycle. But the 99% of product strands that did not contain an error will also be amplified each cycle. Meaning, your error rate in your product strands will just be the error rate of the polymerase -- 1 in 10,000 bases, 1% of the amplicons in this case.
Don't get me wrong: I have sequenced PCR products. The error rate is way higher than 1 in 10,000 bases.
So what is wrong with my logic?
--
Phillip
Use as few PCR cycles during library construction as possible.
One can imagine various rationales behind this one.
- Any bias in the PCR process becomes more pronounced as more cycles are performed.
- If your library is very small (eg, 1 million amplicons) then PCR amplifying it to 1 billion amplicons serves little purpose. You will just end up sequencing those original 1 million amplicons and average of 1000 times each.
- A PCR polymerase has an inherent error rate. The more product strands created, the more errors introduced.
But is #3 even true? Before delving into it, I would like to exclude issues having to do with overrunning the supply of reactants in the PCR. If the amount of final product approaches the total supply of dNTPs in the reaction, I can easily imagine higher levels of misincorporation.
By "errors" here, we mean errors per total bases, right? If the PCR polymerase used is like Taq polymerase it likely has an error rate of about 1 in 10,000 bases polymerized.
Templates:
One thousand 100 base amplicons.
The question: will the error rate per amplicon be higher after 20 cycles than it was after 10 cycles?
Again, presuming reactants are not limiting and each cycle is 100% efficient -- that is, doubling the number of amplicons: After 10 cycles there will be 1 million (2^10 * 1000) amplicons (~0.1 pg of DNA). After 20 cycles there will be 1 billion (2^20 *1000) amplicons (~0.1 ng of DNA).
Doesn't intuition tell us that after 10 additional cycles the errors will have compounded and the overall error rate we might detect by sequencing 1000 of the amplicons will have increased?
Ignoring indels, I don't see this is the case. Sure, Taq polymerase would tend to misincorporate a base in 1% of the product strands that it creates, and that erroneous template will then be amplified each cycle. But the 99% of product strands that did not contain an error will also be amplified each cycle. Meaning, your error rate in your product strands will just be the error rate of the polymerase -- 1 in 10,000 bases, 1% of the amplicons in this case.
Don't get me wrong: I have sequenced PCR products. The error rate is way higher than 1 in 10,000 bases.
So what is wrong with my logic?
--
Phillip
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