Sample Prep and Labeling
IrysPrep kits provide all the reagents needed to label your DNA at specific sequence motifs for imaging and identification in IrysChips. There are a variety of techniques that are useful for labeling DNA. Some are based on enzymatic reactions; others are based on labeling with sequence-specific small molecules. A preferred method we are making available uses a nicking endonuclease to create a single-strand cut in the long DNA molecules at a specific recognition site, wherever it occurs in the genome. Fluorescently labeled nucleotides are then incorporated by repairing the single-strand nicks with a polymerase.
These labeling steps result in a uniquely identifiable sequence-specific pattern of labels to be used for de novo map assembly or for anchoring sequencing contigs. We anticipate commercialization of kits using other labeling techniques in the future.
Linearization and Imaging
A labeled DNA sample is pipetted onto the IrysChip in one or both flowcells. The Irys System controls the movement of DNA in the flowcell electrophoretically. A gradient of micro- and nano-structures, upstream of the IrysChip’s NanoChannels, unravels DNA in solution and overcomes the entropic barrier to confine chromosomal-length nucleic acids inside the NanoChannels.
The current is transiently turned off and the molecules in solution are stationary and stretched uniformly. At this point imaging occurs, capturing high-resolution, single-molecule images of the labeled DNA that contains sequence motifs along hundreds of kilobases to longer than a megabase, in a single contiguous molecule. Once imaged, the molecules are flushed and the process is repeated, allowing imaging of several gigabases of DNA per hour.
The nanofluidic environment allows molecules to move swiftly through thousands of parallel channels simultaneously, enabling high-throughput processing to build a more accurate genome map.
This approach reveals meaningful biological information that is often disrupted or completely lost when molecules are sheared, including:
- Architecture of the order and arrangement of functional regions, despite the presence of repetitive elements that confound other technologies
- Context precisely outlining the relationships between elements
- Interactions among individual or collections of genome elements by direct measurement rather than inference
- Epigenetic modifications occurring across the genome
De Novo Genome Map Assembly
Raw image data of labeled long DNA molecules are converted to digital representations of the motif-specific label pattern. These data are then assembled de novo using IrysView data analysis software to recreate a whole genome consensus map of the original genome. Having a genome map in hand enables a variety of analyses, including sequence finishing and structural variation detection.