Question: why we still need PCR before NGS even when there are brige amplifications for signal reading in Ilumina ?
gravatar for wangyi2412
5.9 years ago by
wangyi2412220 wrote:

For genome sequencing or exome sequencing, especially in practice, there alway needs to do PCR before sequencing. But during sequencing, on the machine, there is already the bridge PCR to amplificate signals; and PCR should be avoided as much as possible because it could introduce PCR duplicate bias to the following analysis.

So, why there needs PCR before NGS? Thank you very much!

ADD COMMENTlink modified 5.9 years ago by Brian Bushnell17k • written 5.9 years ago by wangyi2412220
gravatar for Brian Bushnell
5.9 years ago by
Walnut Creek, USA
Brian Bushnell17k wrote:

You don't need to do PCR prior to sequencing, and it's best not to; when possible, you can get better results by starting with the amount of DNA required.  However, the machines need a minimum amount or concentration of DNA in order to load efficiently.  If you do not have enough DNA - for example, if you are pulling it from a single fly leg, or a single cell - then it should be amplified until you do.

The two amplification steps are unrelated - one is for increasing the signal strength, and one is for stoichiometry for efficient chemical reactions and deposition on the flow cell.

ADD COMMENTlink modified 5.9 years ago • written 5.9 years ago by Brian Bushnell17k
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