Why does the read depth vary across the sequence data for my custom gene?
Plasmid DNA is fragmented prior to sequencing. Next, the fragmented DNA must be converted to a library that is compatible with the NGS sequencing platform, which is done using transposases to randomly add sequencing adapters to the fragments. While this process is somewhat random, it is influenced by the structure and composition of the sequence. Similarly, the success of individual reads within the run is also influenced by structure and sequence. Highly structured areas, areas with extremes in GC, and other complexities tend to affect read depths in areas where they occur.