Quick notes about PCR amplification for Barcode sequencing
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Several guidelines
Guideline for a general reference：
2014-Measuring the activity of protein variants on a large scale using deep mutational scanning
Having too many PCR amplification cycles may not be very good, because
1) Error increases linearly with the number of cycles under usual conditions, (but the product would increase exponentially).
2) Over amplification towards the end of PCR may have reduced accuracy.
For actual amplicon sequencing, the secondary peak will be closer due to smaller bubbles.
Figure 2. My previous amplicon sequencing bubble product. The left and right ones are the lower and upper marker, the correct size should have been 231. Note that the controls are pretty wide, too. Overamplification is also obvious from the intensity of the two peaks.
To avoid bubble product, either decreasing your initial input and decrease the number of PCR cycles (or if you use a higher temperature, with lower amplification efficiency, there would also be less bubble product... but why not just reduce the number of cycles?). Suggested cycle 15 cycles, or 20 cycles. Note that it is also subject to the original concentration of the target segment (whether it is highly expressed gene RT product or just a genomic fragment).
The number of cycles used is related to the PCR efficiency (affected by temperature, enzyme, primer design, concentrations of the template, etc), product at the beginning.
Below is an example of 25 cycles with the PCR efficiency is not quite high (This is the result of a different bioanalyzer machine, always pay attention to how wide your control peaks are).
Note about PCR amplification error of different enzymes
From my personal observation, Kapa HiFi is not as high as they claimed in the manual, which is in accordance with the following paper...
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5218489/pdf/pone.0169774.pdf
Several guidelines
Guideline for a general reference：
2014-Measuring the activity of protein variants on a large scale using deep mutational scanning
Having too many PCR amplification cycles may not be very good, because
1) Error increases linearly with the number of cycles under usual conditions, (but the product would increase exponentially).
2) Over amplification towards the end of PCR may have reduced accuracy.
About the bubble product
Overamplification of PCR may cause bubble product to form, resulting in higher error rate but won't cause problems for sequencing (such as clustering, because denatured libraries have complete adapters on either side for sequence).
Figure 1. Illumina bubble product example
To avoid bubble product, either decreasing your initial input and decrease the number of PCR cycles (or if you use a higher temperature, with lower amplification efficiency, there would also be less bubble product... but why not just reduce the number of cycles?). Suggested cycle 15 cycles, or 20 cycles. Note that it is also subject to the original concentration of the target segment (whether it is highly expressed gene RT product or just a genomic fragment).
The number of cycles used is related to the PCR efficiency (affected by temperature, enzyme, primer design, concentrations of the template, etc), product at the beginning.
Below is an example of 25 cycles with the PCR efficiency is not quite high (This is the result of a different bioanalyzer machine, always pay attention to how wide your control peaks are).
Note about PCR amplification error of different enzymes
From my personal observation, Kapa HiFi is not as high as they claimed in the manual, which is in accordance with the following paper...
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5218489/pdf/pone.0169774.pdf
I think Q5 is the best option unless it turned out to be not so...
Compare with circularized plasmid
One question I get asked about is why is this form of bubble product traveling slower than the linear product, while we know that "the cut plasmid (ie elastic band) is "longer" than the uncut one" or put it another way "uncut plasmid will appear on the bottom of gel while linear will appear top of the gel". But here, the bubble product seems to be longer.
ReplyDeleteI think this two forms are not directly comparable. The bubble product is a mixture of paired and unpaired region but a circularized plasmid is something else.
I have added an illustration to the end of the post.