Abstract: Cyclone Rolling Circle Amplification (CRCA) based next-generation sequencing (NGS) using a multiple primer rolling circle amplification (RCA) reaction is generated using two or more tandem primers from the same strand on different locations of library adaptor regions of double stranded enriched targeted polymerase chain reaction (PCR) library products. This process allows multiple initiation and syntheses of the RCA reaction by an enzyme on the same circular template molecule, which is beneficial since the two or more primers complement each other in generating uniform amplification of the target circle population. Also, a method for keeping DNA nanoballs (also known as rolonies) compact, or more compact, and for pre-priming rolonies before sequencing to eliminate the hybridization of a seqeuncing primer after seeding the rolonies, and a Rolonies rolling circle amplification (RCA) based next-generation sequencing (NGS) using a dual Rolonies primer approach named REPLI-Rolony.