Ligation of Illumina adapters to custom adapters

[ale]

I would like to create libraries with the following protocol but the second adapter ligations seems not to work:

1. MseI digestion of genomic DNA
2. Custom adapter ligation with MseI overhang on one side and A-tail on the other end
3. Ligation of Illumina Y-adapters to sequence the library on the MiSeq (after step 9 in the attached figure)

While the first ligation appears to work, the second ligation does not at all. I tried then to End-repair and A-tail the fragments (with the first custom adapter attached) but when I run PCR with Illumina primers after that, there is still no PCR product of the fragments.

What could here go wrong? T custom adapters have phosphorylated 5'-ends and A-tails at the 3-end that is bound to the next base with a phosphorothioate bond (small s in the attached drawing) in order to prevent exonucleases from cutting off the A-tail.

I appreciate any kind of help.

[nucacidhunter]

Your workflow seems OK and does not require end repair and A tailing. End repair may not work because of the phosphorothioate bond. If you are using ligation mix and adapters from Illumina kit with 20-50 ng of MseI adapter ligated DNA, it should work.

[ale]

Dear nucacidhunter,

I will try the second ligation a last time:
1. with the first custom adapters ligated
2. only with the end-repaired and A-tailed restriction cutting sites

This should show me if the problem is the second ligation or the characteristics of the first custom primers