Image extremely long, high-molecular-weight DNA for analysis with Saphyr’s streamlined workflow.
Bionano sample preparation kits provide everything needed to isolate extremely high-molecular weight-DNA from fresh or frozen blood, cultured cells, and plant and animal tissues.
Unlike standard DNA isolation methods such as precipitation, column, or bead-based methods, which yield reads too small and fragmented for assembly, Bionano’s sample prep method produces the large molecules, greater than 150kbp, needed for assembly.
Detailed protocols for isolating high-molecular-weight DNA are provided to users. These protocols are based on the isolation of cells or nuclei in an agarose matrix, where DNA purification takes place while the molecules are stabilized in agarose. By the end of the purification process, the agarose is digested and molecules up to chromosome arm lengths in size are released.
The Bionano DNA labeling kit provides the reagents needed to label DNA at specific sequence motifs for imaging and identification. These labeling steps result in a uniquely identifiable sequence-specific pattern of labels to be used for de novo map assembly.
Bionano has two labeling chemistries, one based on nicking endonucleases and a second using our newest Direct Labeling chemistry. In nickase based labeling, a nicking endonuclease creates a single-strand nick in the long DNA molecules at a specific recognition site, wherever it occurs in the genome. Then, fluorescently labeled nucleotides are incorporated and single-strand nicks are repaired with a polymerase and ligase.
When using our Direct Label and Stain kit, the sequence motif is labeled fluorescently in a single direct enzymatic reaction.
Linearization and Imaging
A labeled DNA sample is pipetted onto the Saphyr Chip™ in one of the flowcells. Saphyr electrophoretically controls the movement of DNA in the flowcell. A gradient of micro- and nano-structures, upstream of Saphyr Chip’s NanoChannels, gently unwinds and guides DNA into the NanoChannels.
Saphyr Chip’s NanoChannels allow only a single linearized DNA molecule to travel through while preventing the molecule from tangling or folding back on itself. The nanofluidic environment allows molecules to move swiftly through hundreds of thousands of parallel NanoChannels simultaneously, enabling high-throughput processing to build an accurate Bionano genome map.
Once the DNA is stretched inside the NanoChannels, the high-resolution Saphyr camera images them. Long molecules spanning beyond a field of view are stitched together. Once imaged, the molecules are flushed and the process is repeated, enabling imaging of >25Gb of DNA per hour.
DE NOVO GENOME MAP ASSEMBLY
Once raw image data of labeled long DNA molecules is captured by the Saphyr instrument, it is converted into digital representations of the motif-specific label pattern. Bionano Solve™ data analysis software then assembles the data de novo to recreate a whole genome map assembly.
Bionano genome maps enable a variety of analyses, including hybrid scaffolding and structural variation detection. Next-Generation Sequencing (NGS) analyses often rely on alignment of short reads to a reference to infer the underlying genome structure. With this strategy, the ability to decipher regions where the reference itself is incorrect or significantly different from the genome of interest is lacking. Bionano assemblies are not guided by a reference, allowing for unbiased reconstruction of the genome structure.