I want to add inline barcode within custom adapters for multiplexing sequencing. The sequencing lab manager suggested me just adding the 4-6bp indexes on one end of adapter. Thus two oligos are not complimentary on the end to form a Y-shape any more.
She explained to me that they didn't anneal them and just mixed equimolar amounts for ligation. The one with 5'Phos goes on 3'-end of library fragment. The non-phosphorylated one goes on 5'-end of fragment.
I don't know if someone here did this before for a regular paired-end library? But I can't figure out how the DNA ligase works on this condition.
She explained to me that they didn't anneal them and just mixed equimolar amounts for ligation. The one with 5'Phos goes on 3'-end of library fragment. The non-phosphorylated one goes on 5'-end of fragment.
I don't know if someone here did this before for a regular paired-end library? But I can't figure out how the DNA ligase works on this condition.
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