- PgmNr 2598/W: Bionano Prep SP isolates high quality ultra-high molecular weight (UHMW) genomic DNA to improve research of cancer and undiagnosed disorders.
H. Sadowski; C. Proskow; A. Files; K. Pham; D. Carroll; H. Barnes; Y. Zhang; G. Pljevaljcic; M. Borodkin
Affiliation: Bionano Genomics, San Diego, California, USA
Optical mapping of genomic DNA on the Bionano Genomics Saphyr® system for genome assembly or structural variation detection relies on starting with UHMW DNA. To achieve this, protocols using agarose plugs have been the gold standard. The “plug lysis” method is extremely robust, but it is also labor intensive, difficult to automate, lengthy and expensive. To address these shortcomings, we coupled solution-based lysis with a purification step that leverages a novel process to bind, wash and elute UHMW genomic DNA. This entire protocol can be conducted in less than 4 hours on a batch of 6 samples, allowing 12 samples to be processed in one day. The eluted material is ready to use by day 2 and contains high quality DNA that is clean enough for the direct label and stain (DLS) protocol. The resulting single molecule quality metrics of this labeled DNA on a Saphyr Chip® are comparable or exceed the metrics for labeled DNA isolated by the traditional plug lysis protocol. We have previously validated protocols for fresh/frozen human blood (EDTA) and cultured cells and released the Bionano Prep SP kit that provides virtually all of the required reagents and consumables. For many samples, the addition of DNA stabilizer has allowed us to achieve average labeled DNA centers of mass (COM) N50s of ≥ 280 kbp, with a significant proportion of Mbp-sized molecules. Unlike plug lysis, the SP protocols are clearly automatable, providing a solution for researchers needing to purify DNA from hundreds to thousands of individuals per year.
We have continued to expand the number of validated SP protocols for new sample types including fresh/frozen heparinized human blood and bone marrow aspirates (BMA)s. The new protocols also include; the addition of DNA stabilizer to the heparinized bloods/BMAs before they are frozen, to increase the size of isolated DNA (COM N50s ≥ 300 kbp), and cell straining upon thawing, to avoid clots and insoluble aggregates before cell counting. We have also developed working SP protocols for small amounts (5-15 mg) of animal and human tissues including tumor biopsies. These SP protocols are significantly shorter and less labor intensive than our previously released plug lysis protocols, while QC metrics of the SP isolated genomic DNA are comparable or exceed the metrics of genomic DNA isolated by the plug lysis protocols.
PgmNr 2598: Bionano Prep SP isolates high quality ultra-high molecular weight (UHMW) genomic DNA to improve research of cancer and undiagnosed disorders.
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