I do rolling circle amplification (RCA) on my ligated PCR products prior to Nanopore sequencing. After using enzymatic and mechanical (covaris g-tube) fragmentation of the RCA products, my sequencing attempts generated severely short reads (
This should be possible without fragmentation. ON's minION device has been successful with their 1d kit after de-branching RCA products with T7 endonuclease.
Erina Rader so you are suggesting not to skip the enzymatic reaction, as it is essential prior to the library prep?
Hi Bilgenur Baloglu, did you try without enzymatic reaction?
Last time I used the rapid kit because was the quicker was of cutting + library prep at the same time. But because there is a branching strucutre from the RCA I have the same question as you: would the sequencing work without fragmentation?
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