DNA was isolated from blood of a six-month-old male FHM using the Blood and Cell Culture DNA Isolation Kit (Bionano Genomics, San Diego, CA). Nucleated fish blood was embedded in agarose plugs for titration in volumes of 1 µL, 3 µL, 8 µL, 20 µL, and 50 µL. A proteinase K digestion was performed, followed by additional washes and agarose digestion. The agarose plugs were sent to the McDonnell Genome Institute (MGI) Washington University for processing and analysis.
From this point, the DNA was drop dialyzed and allowed to equilibrate at room temperature for two days. The DNA samples were assessed for quantity and quality using a Qubit dsDNA BR Assay kit and CHEF gel. A 750ng aliquot of DNA was labeled and stained following the 'Bionano Prep Direct Label and Stain'(DLS) protocol. Once stained, the DNA was quantified using a Qubit dsDNA HS Assay kit and run on the Saphyr chip. During the Saphyr run, the DNA were processed through a series of micro and nanochannels to prevent the linear DNA from folding back on itself. Once the DNA is linearized into the nanochannels, the data were imaged and captured by the Saphyr instrument.
From this point, the DNA was drop dialyzed and allowed to equilibrate at room temperature for two days. The DNA samples were assessed for quantity and quality using a Qubit dsDNA BR Assay kit and CHEF gel. A 750ng aliquot of DNA was labeled and stained following the 'Bionano Prep Direct Label and Stain'(DLS) protocol. Once stained, the DNA was quantified using a Qubit dsDNA HS Assay kit and run on the Saphyr chip. During the Saphyr run, the DNA were processed through a series of micro and nanochannels to prevent the linear DNA from folding back on itself. Once the DNA is linearized into the nanochannels, the data were imaged and captured by the Saphyr instrument.