Next-generation sequencing has become an essential tool in molecular biology that has been successfully applied to a broad variety of experimental approaches. While several platforms for next-generation sequencing exist, the most commonly used approach is sequencing-by-synthesis, implemented on Illumina's Genome Analyzer II (GAII) and HiSeq2000 systems. A key constraint of these sequencers is the need to run multiple lanes of samples with identical parameters as part of a single flowcell. Here, we present a series of modifications to the Illumina Genome Analyzer II, along with a script generating tool, that allow users to run the GAII in a lane-by-lane manner. Any number of lanes can be run at one time. Repeated use of the same flowcell on multiple sequencing runs does not appreciably reduce the intensity, cluster density, or accuracy of the run. These modifications will enable smaller-scale experiments with unusual design parameters to be run routinely on the GAII.