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Plasmid DNA is fragmented and converted into a sequencing library before NGS analysis. Although adapter insertion is partly random, sequence structure, GC content, repetitive regions, and other complexities can affect local read depth and read quality.
Even clonally pure samples rarely show 100% identity in every NGS read. Sequencing and analysis errors are typically about 0.1% to 0.01% and may be locally elevated in difficult regions. Trace misalignment and small subpopulations of variants arising during E. coli propagation can also contribute.