Bionano prep dls labeling kit

For POC negative for MCC, ~15 mg of POC section was used to isolate ultra-high molecular weight (UHMW) molecules using the Bionano Prep SP DNA Isolation Kit. Subsequently, the Bionano Prep DLS Labeling Kit was used to fluorescently label long molecules at specific sequence motifs throughout the genome. The labeled DNA was loaded onto Saphyr chips for linearization and imaging in massively parallel nanochannel arrays. The observed unique patterns on single long DNA molecules were used for de novo genome assembly and structural variant calling via the Bionano Solve pipeline (version 3.6).

DNA was extracted from tissue using the Bionano Prep SP Tissue and Tumor DNA Isolation Kit (Bionano, San Diego, USA) and a Tissue Ruptor (Quiagen) targeting a final concentration of 50–120 ng/ $\mu$ L. DLE-1 labeling was done using the Bionano Prep DLS Labeling Kit with final concentration targeted at 4–12 ng/ $\mu$ L. Data collection was performed using Saphyr 2nd generation instruments.

OGM was performed on the 4 cases. The brief experimental procedures were as follows, briefly: Ultra-high molecular weight (UHMW) gDNA was isolated from peripheral blood following the manufacturer's guidelines (Bionano Prep SP Frozen Human Blood DNA Isolation Protocol, Bionano Genomics, San Diego, CA, USA). Thereafter, the direct label and stain (DLS) technique was used according to the manufacturer’s instructions (Bionano Prep DLS Labeling Kit; Bionano Genomics, San Diego, CA) to label the DNA. The direct labeling enzyme 1 (DLE-1) reaction was carried out using 750 ng of high-molecular-weight gDNA to tag a specific 6-bp sequence (CTTAAG). Subsequently, the fluorescently labeled gDNA molecules were loaded on Saphyr chips for linearization and sequential imaging in massively parallel nanochannel arrays. De novo assembly and structural variant (SV) calling were performed via a de novo assembly pipeline through Bionano Access v1.2.1 (Bionano Genomics, San Diego, CA) and compared with Genome Reference Consortium GRCh38 (hg 38). Variant types such as insertions, deletions, inversion breakpoints, translocation breakpoints, and CNVs were detected with this pipeline [22 (link), 23 (link)].