paired end sequencing, sequence distance

Question

[tonja.r]

Hello,

I am a bit confused about the sequencing in paired end reads, single reads and mate paired reads.

I know the difference between the protocols and why matepairs map <---- -----> and they have a much longer distance between them.

​When performing sequencing on single reads, we sequence till the second adapter, meaning till the end. So, we sequence the entire fragment. With paired end reads as well as with mate paired reads we know the fragment sizes from our fragmentation step and we define how long the reads are gone be. For instance, we stop sequencing when our read length,let say, is about 80pb.

From the very beginning we know that the fragments in ME protocol are larger, so if we sequence only 80 bases towards the fragment beginning points (biotin labels), we have a large distance between them. With the PE we sequence till the ends of the fragments but we stop at some point.

Why cannot we perform PE sequencing on large fragments (as we do with ME)? We will sequencing till the end of the fragments but we can stay stop after 80bases and at the end we will have the same distance as with ME.

Answer 1

[thackl]

I'm sorry, I'm not entirely sure, what you are asking. If I understand you correctly, you want to sequence long fragments (>1kb) PE style on a Illumina machine. The Problem here is that for fragments of this length, the bridging and amplification on the flowcell will not work.

MP fragments on the machine aren't actually long fragments, but only the region of the circularized fragment, comprised of both of the fragment ends and the biotin label.

[Devon Ryan]

Nicely put!

Answer 2

[Devon Ryan]

You don't typically sequence the entire fragment with single-end sequencing. You sequence 75, 100, 150, etc. bases from one end. This is exactly the same as paired-end sequencing. Paired-end sequencing starts as single-end sequencing and then flips the fragments after finishing and starts sequencing from the other end.

Mate-pair sequencing doesn't work how you seem to think it does. The only difference between mate-pairs and paired-end is in the library preparation. The actual sequencing is the same (i.e., mate-pairs are paired-end sequences, just library is made differently). Below is a little graphic showing how mate-pairs work:

So you can see at step 4 you have a standard fragment that's used for paired-end sequencing.