The following is an overview of each cycle in the optical sequencing process.
Step 1: DNA barcoding<br />
Cells are lysed to release genomic DNA.  These DNA molecules are untangled, placed onto optical mapping surface containing microfluidic channels and the DNA is allowed to flow through the channels.  These molecules are then barcoded by restriction enzymes to allow for genomic localization through the technique of optical mapping.  See the above section on "Technology" for those steps.
Step 2: Template nicking<br />
DNase I is added to randomly nick the mounted DNA molecules.  A wash is then performed to remove the DNase I.  The mean number of nicks that occur per template is dependent on the concentration of DNase I as well as the incubation time.
Step 3: Gap formation<br />
T7 exonuclease is added which uses the nicks in the DNA molecules to expand the gaps in a 5–3 direction. Amount of T7 exonuclease must be carefully controlled to avoid overly high levels of double-stranded breaks.
Step 4: Fluorochrome incorporation<br />
DNA polymerase is used to incorporate fluorochrome-labelled nucleotides (FdNTPs) into the multiple gapped sites along each DNA molecule.  During each cycle, the reaction mixture contains a single type of FdNTP and allows for multiple additions of that nucleotide type.  Various washes are then performed to remove unincorporated fdNTPs in preparation for imaging and the next cycle of FdNTP addition.
Step 5: Imaging<br />
This step counts the number of incorporated fluorochrome-labeled nucleotides at the gap regions using fluorescence microscopy.
Step 6: Photobleaching<br />
The laser illumination that is used to excite the fluorochrome is also used here to destroy the fluorochrome signal.  This essentially resets the fluorochrome counter, and prepares the counter for the next cycle.  This step is a unique aspect of optical sequencing as it does not actually remove the fluorochrome label of the nucleotide after its incorporation. not removing the fluorochrome label makes sequencing more economical, but it results in the need to incorporate fluorochrome labels consecutively which can result in problems due to the bulkiness of the labels.
Step 7: Repeat steps 4–6<br />
Steps 4-6 are repeated with step 4 using a reaction mixture that contains a different fluorochrome-labeled nucleotide (FdNTP) each time.  This is repeated until the desired region is sequenced.