PolyA on flowcell

[biochembug]

Hi Friends

If I plan to clone RNA near its 3' end and somehow select just the 3' ends. What do you think about clusters when sequencing the A tail. I am planning to sequence from the back-end of the RNA. Will the clusters be unique or because every spot will glow for A (or T) it might confuse with the clusters?

Does anyone have idea whether we can place a barcode before the insert and read it as a part of insert? Any alternatives?

Please share your experiences and suggest some other ideas?

Biochembug

[HESmith]

Accurate cluster calling requires sequence diversity during the first 4-5 cycles. You can design your primers to sequence from the non-A end, or engineer 5mer barcodes with equal representation before the polyA.