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I recently learned about the PCR testing and its idea of using primers to replicate the DNA or RNA. By this I assume that we need to know the sequencing of the DNA to make our primers. If that is true; then how did we do the COVID PCR in the early days of the pandemic? Is it possible to do the sequencing on a sample without amplifying it first with PCR?

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2 Answers 2

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Indeed, one can sequence genome directly without prior amplification - this is usually referred to as shotgun sequencing. Obviously, this is a time-consuming and costly approach. Therefore, once the biomarker of interest is identified, one focuses on amplifying and detecting only this biomarker.

Note also, that there exist generic sequencing primers, which could, with high probability, initiate DNA synthesis for preferred type of organism/virus.

Remark
@bob1 has pointed in the comment that what we call time consuming and costly is relative: e.g., sequencing human genome costs <\$1000 per sample, and takes a couple of hours to run - in this sense it is not too costly to be done in a research lab, but it is still quite costly in terms of everyday testing of millions of people. The cost of commerically available PCR tests for covid-19 is about \$50-\$100.

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    $\begingroup$ These days people just do whole genome sequencing for something like this. It's not particularly costly (<USD$1000/sample), and sequence agnostic - a bunch of blunt-end ligations to get the library prep done, then put it on the machine. ~2h-3d later you get the results, 1h analysis. $\endgroup$
    – bob1
    Aug 31, 2021 at 9:50
  • $\begingroup$ @bob1 I added a clarification, taking into account your comment. $\endgroup$
    – Roger V.
    Aug 31, 2021 at 10:01
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    $\begingroup$ I was more meaning in the context of the OP's question which boils down to "how did we get the initial sequence to design the primers" The answer is: it was done by whole genome sequencing. I've just had a look at the cost of a miSeq run, and it's about \$2000 - you can get 96 samples on that, so about \$20/sample, but not as simple as quick or as automated as PCR can be. $\endgroup$
    – bob1
    Aug 31, 2021 at 10:10
  • $\begingroup$ @bob1 I understood the question as asking whether we could sequence without amplification. But to identify the specific sequences corresponding to the virus one probably had to sequence everything that was in the sample - this is probably what you mean. $\endgroup$
    – Roger V.
    Aug 31, 2021 at 10:55
  • $\begingroup$ You can't sequence 96 full human genomes on a MiSeq. I don't think you can do one. $\endgroup$
    – swbarnes2
    Aug 31, 2021 at 16:40
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As an extension of Roger Vadim's answer and bob1's comment, note that preparation of DNA libraries for shotgun sequencing often does involve PCR, but the PCR does not use primers that are specific to the sequence of interest. Rather, the primers are either random or specific to so-called adapter sequences that are appended to the ends of genomic DNA fragments in a mostly random fashion. An example of such a workflow would be...

  1. Obtain genomic material from a pure culture of your organism / virus of interest
  2. For genomic DNA, fragment genomic material to a desired insert size. For RNA genomes, use random primers to carry out reverse transcription.
  3. Treat your DNA/cDNA libraries with a pool of enzymes that makes the fragment ends amenable to ligation of adapters. This process is often called "blunting and A-tailing".
  4. Ligate adapters to the ends of treated fragments.
  5. PCR amplify your DNA libraries using primers that recognize the specific sequence of the adapters. This creates DNA fragments that contain genomic DNA flanked by the sequences of your choosing. Often, this appended DNA will include sequences needed by your sequencing platform (read up on bridge amplification in context of Illumina sequencing) as well as short "barcodes" that identify your DNA library as being associated with a specific biological sample.

Once you've got your "reads" from the DNA sequencer, you can use those reads to construct a complete genome in a process known as de novo assembly. If everything goes right, you now have a genome against which you design primers for faster, targeted PCR detection.

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