Why Use Pcr Instead Of Cheaply Multiplying The Data Set Afterwards With Software?

Question

[Herpson]

I found out that Bridge PCR is used to multiply the reads that are fed into the sequencer. I am struggling to see the advantages of

1. having duplicate reads in my data set
2. performing this chemical, surely expensive procedure instead of just "repeating" a simple sequenced data set multiple times

Why would you sequence the same piece multiple times? Why don't I just sequence it once?

[Damian Kao]

Most amplification steps are done for the sake of sensitivity.

Answer 1

[Devon Ryan]

Bridge PCR is used to generate clusters of the same sequence on the flow cell. Without this, it's likely that the camera could resolve the subsequent sequencing reactions. Have a look at this video from Illumina, which is helpful.

Answer 2

[Emily]

You don't PCR it because you want lots of copies of the data, you PCR it to make sure you get sequence coverage - not every sequencing read will be any good.

Answer 3

[swbarnes2]

Bridge PCR is what's done on the flow cell. You have to do that, to amplify the single DNA strand that sicks to the flow cell into a cluster of identical strands, so that the fluorescence can be detected.

As to PCR before the DNA is added to the flow-cell, there are PCR-free protocols, but in general, you will not have enough suitable, properly adapted sequences to cover the flow-cell if you do not amplify them first. The other virtue of that PCR step is because your PCR primers are on the adapters, you know you are only amplifying DNA sequence that will stick to the flow-cell. So any other DNA that's been carried along will be out-competed by the stuff you want. And then when you quantify, you know that your DNA is virtually all stuff that will stick to the flowcell.

Running the instrument costs a lot of money in reagents, and time. It's not a trivial thing to rerun a sample because you didn't have enough reads.