Illumina Paired-End Sequencing On Samples Of Different Sizes

Question

[Sakti]

Once again, asking the wisemen of Genomics :)

My question is the following:

I want to sequence the start and end bases of the PCR products produced by a certain pair of oligos. The situation is that these oligos generate products of different sizes (from 200 bp and up to 9Kb). As far as I know, Illumina always makes a gel-size selection for the DNA to be sequenced, but this is definitely not what I want (I don't want to bias my detection only to certain products!)

Can I do my paired-end sequencing using all of my PCR products, or will there be a bias to report shorter products once the sequencing is performed??

Thanks!!

Answer 1

[Gww]

If you try and ligate adapters onto 9kb fragments and sequence them you will have two issues.

1) The fragments may be too large to properly work in a flow cell when you perform the paired-end sequencing.

2) You will only sequence the ends of the fragment. (I am not sure if this is your goal).

You could fragment the PCR products and then sequence the fragments but you may not be able to reconstruct the original fragments doing this.

EDIT:

From your comment it seems like you are interested in doing paired-end tag sequencing. There is a paper about that here and wikipedia has an article on it here. I don't think you can directly sequence large fragments as you will probably have issues in the ligation and cluster generation steps.

[Sakti]

Hi, yes, in principle I'd like to only get the ends of the fragments, so I'm not worried about not getting the full sequence of the PCR products. Thanks

[Gww]

I added a comment that may help then

[Sakti]

Thanks! I'm already checking it, will let you know how it goes :)