All cells contain a complete copy of the organism's DNA, the genetic blueprint of life, packaged into discrete units called chromosomes. Since new cells need a copy of the genetic material, the chromosomes must be completely and accurately replicated before the cell can divide. This requires the process of DNA replication to start at thousands of sites across the chromosomes - called DNA replication initiation sites. Our project aims to determine the location of replication initiation sites in human cells. This is important because the location and distribution of replication initiation sites have been implicated in causing human diseases such as cancer. It has been challenging to identify DNA replication initiation sites in human cells, because there is a lot of variability between cells. To date, most experiments have used the average from millions of cells, but this hides the variability between cells. We have developed a novel technology that identifies DNA replication initiation sites on thousands of single molecules that each originated in a single cell. Our approach can also identify specific sequences that are challenging to copy. This will allow us to test the hypothesis that some sites, thought to be involved in replication initiation, in fact are sites that impede DNA replication. Distinguishing between sites that initiate versus impede DNA replication will be crucial in understanding the causes of genetic instability that underlie some human diseases.