What is the longest fragment for Illumina Mate pair end seq library?

[ychang]

Hi, every, I want to prepare a library for mate pair end seq, the special aspect of the library is that I want the length of the fragment as long as possible, say, 10kb or even 20kb. Do you think the circularization efficiency will reduce with the increase of the length?

Thanks!

[tonybolger]

Hi, every, I want to prepare a library for mate pair end seq, the special aspect of the library is that I want the length of the fragment as long as possible, say, 10kb or even 20kb. Do you think the circularization efficiency will reduce with the increase of the length?

Thanks!

You can push the illumina protocol to at least 12Kb, using the 10Kb semi-official protocol but the quality heads downwards.

One alternative is to use the 454 linker approach, but ligate illumina adapters on the result - this should work up to 20Kb, and can possibly be pushed up to 65Kb with some tweaks, according to this

Another alternative is the fosmid approach, detailed here

I would be interested to know if any of these approaches work for you

[monad]

Make sure that your starting input DNA is enough. Longer the insert, you will need a lot more input DNA. We are talking about ~10ug for end repair for ~12kb insert mate pair, so starting material is whatever the recovery rate after shearing.

[Liting]

Hello，Monad.I have some problems and hope for your help.Thank you.I used 20ug DNA for end repair for 6kb insert mate pair.The protocal is :the DNA with cycling adaptors,size selection after nebulization,circularization & linear Digestion and nebulized again,then library immobilization for paired-end library construction.I used agarose gel electrophoresis to check the size of library. It's bigger than 100bp.The result is abnormal.I can't figure out why.Please give me some advices.Thank you very much!

[andyding]

What about whole genome amplification before library prep?
Cheers.

Make sure that your starting input DNA is enough. Longer the insert, you will need a lot more input DNA. We are talking about ~10ug for end repair for ~12kb insert mate pair, so starting material is whatever the recovery rate after shearing.