PolyA on flowcell

biochembug · 09-22-2011, 05:56 AM

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 · 09-22-2011, 09:05 AM

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.

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09-22-2011, 05:56 AM	#1
biochembug Member Location: New York Join Date: Mar 2011 Posts: 26	PolyA on flowcell 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

09-22-2011, 09:05 AM	#2
HESmith Senior Member Location: Bethesda MD Join Date: Oct 2009 Posts: 509	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.