size of DNA fragments for library preparation.

[jb1]

Can anyone let me know if I have this right?

For the Illumina ChIP kit library preparation, there is a size selection step following adapter ligation for fragments +/- 25bp around 200bp.

The adapter sequences for genomic library prep are 92nt (from another thread on this forum).

So this means that the only fragments included in the library will be 108bp +/-25.

With sonication or enzymatic fragmentation of DNA, most people seem to acheive a mean fragment size of around 150-300bp.

Wouldn't this make a less than ideal library??

[huguesparri]

Hello jb1,
Size selection on a ChIP Seq library preparation occurs before the PCR.
Adapters sequence at this step are only 68nt long (34*2). So your DNA fragment of interest is 132 +/-25 nt long.
As you can see, it's close to the maximum enrichment size of your fragmentation.
Moreover, even if at the end of the sonication the maximum enrichment size is beetween 150 and 300 pb (and even higher), there is a lot of lower size DNA in the smear.
In the end, as long as your fragmentation is random, libraries generated this way are ok and reprensentative of your total ChIPed material.

[jb1]

Thanks for your reply, that really clears things up. I will aim for fragments of 132bp on sonication!

[huguesparri]

You can also try to cut your gel at higher size (like 250+/-25) to be sure to be in the enriched region.
It shouldn't affect the sequencing process.