How the first sequencing template removed in pair end sequencing

Question

[octpus616]

Hi

I want to know the details of pair-end sequencing, especially how the products of the first sequencing are removed.

I understand that pair-end sequencing is a method of DNA sequencing that can simultaneously sequence both ends of a DNA fragment, obtaining more information and higher accuracy. I also know that pair-end sequencing involves three steps: library preparation, amplification and sequencing, and alignment and analysis. However, I am confused about the following points:

How are the products of the first sequencing removed? I know that there are some methods, such as using double-strand specific nuclease (DSN). But if a double-stranded DNA-specific restriction enzyme is used, how can the template for the second sequencing be protected from being destroyed, since it is also a part of the double-stranded DNA?

I would appreciate it if someone could explain these points to me in detail or provide some references for further reading. Thank you very much.

=====update=======

Thanks to @rfran010 's answer, it seems that I have confused the product of the first sequencing with the PCR process in preparation for the second sequencing. The products of the first sequencing should be easily removed because there is no covalent connection.

So the question is, actually, how did the template of the first round be removed here? Through specific restriction enzymes?

[rfran010]

Granted, I do not have any technical expertise and the bulk of my knowledge is from the video here, https://www.illumina.com/science/technology/next-generation-sequencing/sequencing-technology.html

However, I believe your picture is not of paired-end sequencing, but the amplification/cluster generation process. From here, they would cleave the reverse strands (represented in purple-anchored strands) before doing the READ 1 reads. The details of cleavage, I do not know. However, it seems straightforward to use a specific sequence/modification and enzyme that can reliably cleave the region they want to cleave.

The picture here is essentially a standard PCR, no sequencing chemistry applied yet.

Beyond this, the first sequencing/index products could be removed from denaturation since they are not attached to the flow cell. Then, it seems the bridge amplification takes place again, but this time the forward strand is cleaved and the reverse strand is sequenced to produce READ 2.

This is how I understand it, but I'm really not an expert.

[octpus616]

Hi, thanks for your answer, I think I have confused the product of the first sequencing with the PCR process in preparation for the second sequencing, I updated this question.

[GenoMax]

Far as I know the details of chemistry and enzymes that makes all of this possible are obscure/trade secrets. You could try and pore through solexa/Illumina patents but the exact details are likely obfuscated.

[Asaf]

I thought it was an 3'->5' exonuclease.

[octpus616]

The issue here is the lack of exposed double-stranded ends to support the activity of the 3'->5' exonuclease. If a single-strand-specific restriction enzyme is used, it will be difficult to protect the template for the second sequencing.

Answer 1

[rfran010]

Great! It looks like USER enzyme is used to cleave before the first read sequencing and formamidopyrimidine DNA N-glycosylase is used to cleave before the second read sequencing.

These are used since the bound oligos on the flow cell have either a 2-deoxyuridine or 8-oxoguanine modification (modifications that will allow cleavage by the respective enzymes above)

At least that's in 2008. Not sure how they changed their chemistry since then.

https://www.nature.com/articles/nature07517

[GenoMax]

Moving this to an answer since there is a link for a paper. Things could have changed as @rfran010 says but this may be the closest we will get to knowing specifics.