Nanopore sequencing error benchmarking in RCA enriched circular DNA viruses

This project characterizes base level sequencing errors introduced by rolling circle amplification (RCA) and nanopore sequencing in small circular single stranded DNA genomes. A controlled model system was created using the 7.2 kb Enterobacteria phage M13mp18 genome serially diluted into pooled human plasma DNA extracts positive for Anelloviridae to simulate complex viral metagenomic backgrounds. RCA was performed under 1X and 3X shearing conditions to evaluate the effect of fragmentation on read length and error profiles. Sequencing was conducted on the Oxford Nanopore GridION platform using R10.4.1 flow cells and Dorado basecalling. Base level mismatches, insertions, and deletions were quantified using custom alignment parsing tools to determine per base error rates and consensus accuracy across input concentrations and simulated depths. These data establish an empirical benchmark for nanopore sequencing of RCA enriched circular viral genomes and define quantitative thresholds for reliable genome reconstruction in complex metagenomic samples.