TruSeq PCR-free, ligate adapters to amplicons?

[JBKri]

We want to ligate Illumina adapters to already barcoded PCR-products, using a TruSeq PCR-free library kit. Have anyone tried it? Are there any published protocols?

So instead of starting with sheared DNA we will have a couple of different amplicon sizes (~100 bp, ~150 bp). They will be purified with AMpure XP beads and quantified and pooled. Should we then continue with end-repair? I saw elsewhere that this step is needed for for 5' phosphorylation. How much DNA input do we need? The Illumina protocol for shotgun libraries suggest 1 ug (before shearing), which will probably be very difficult to acchieve with our amplicons. Since there will be no two-sided size selection I guess we can use far less, but what is the minimum?
Jon

[nucacidhunter]

I would start from end repair and consider following:

1- TruSeq PCR-free kit input for 350bp insert size is 1 µg.
2- Your fragments are 100-150bp, around 1/3 of 350bp
3- To have equivalent molar fragment input you would need 300 ng.
4- This will be reduced further by considering an approximately 50% loss (never quantified just a guess) during size-selection of standard protocol. So 150 ng should fine.
5- If the yield is not enough or you have less input, couple of PCR cycles should not make any difference as your input are amplicons.

[JBKri]

I would start from end repair and consider following:

1- TruSeq PCR-free kit input for 350bp insert size is 1 µg.
2- Your fragments are 100-150bp, around 1/3 of 350bp
3- To have equivalent molar fragment input you would need 300 ng.
4- This will be reduced further by considering an approximately 50% loss (never quantified just a guess) during size-selection of standard protocol. So 150 ng should fine.
5- If the yield is not enough or you have less input, couple of PCR cycles should not make any difference as your input are amplicons.

Thank you nucacidhunter, good advice as always!