Pair End Sequencing Problem

Question

[jan.shegers]

Dear all,

I have a quick question on pair end sequencing. I used to work with Illumina without the pair end reads and I have dificulties to understand how the pair end reads work.

In the "old" system you removed the opposite strand since that primer had a cleavable site to remove it.. Now how does it work with the paired reads? Do you still remove the opposite strand? if so: how do you "flip" the DNA to read from the opposite side? Or do they not cut one of the primers anymore and sequence 1 strand using 1 primer and the opposite strand with primer 2 ?

Answer 1

[JC]

Recent Illumina pipelines removes the primers in both sequences and many algorithms are aware of the sense of both pairs, you don't need to do nothing, you can map/assemble/whatever-you-do directly from the fastq produced.

Answer 2

[jan.shegers]

Not sure I understand you..

I am not worred about the output, the data.

I am just wondering about how the proces works. In the old system you removed 1 string (the complementary) by cutting 1 adapter.

But in the new system they use pair end and sequence from both sides, but how? I cant imagine they still remove 1 DNA string, sequence the other and than what? How do they sequence the other?

Or do they not remove 1 of the strands and sequence both using 2 different primers?

[JC]

The new protocol use 2 different primers, check this video:

[jan.shegers]

This explains what I already know... its the old system, not the pair end one...

As I told in the previous post: you remove the complementary strands and then you sequence the strands still attached to the flow cell. But how does the pair end thing work?

They remove the strands after the sequencing and then do a new bridge amplification step ? Or?