High-Throughput, High MW DNA Extraction and Size Selection for Long-Read Sequencing.

Long-read sequencing technologies from PacBio and Oxford Nanopore are rapidly becoming the de facto methods for de novo assembly, phasing, and structural variant analysis. These capabilities are predicated on high purity, high molecular weight (HMW) DNA input. However, the gold-standard plug lysis method is tedious and exceedingly slow while conventional spin columns and magnetic beads shear DNA resulting in compromised read lengths. We present novel methods for rapid HMW DNA extraction and library preparation based on our Nanobind magnetic disks. Eachdisk(1-5 millimeter diameter) is covered with a high density of micro- and nanostructured silica that protects DNA from damage and enables high extraction yield and purity. DNA extraction is performed using a simple bind, wash, and elute process that can obtain HMW (300+ kb) and megabase (1+ Mb) DNA in less than 1 hour for most sample types. Automated HMW DNA extractions can be performed using ThermoFisher KingFisher instruments. HMW DNA extraction and sequencing results from a wide variety of sample types including cultured cells, gram-negative and gram-positive bacteria, whole blood, plants, insects, and tissues are shown. PacBio SMRT sequencing on Sequel System typically results in subread length N50s up to 40 kband throughputup to 15Gb per flow cell. Sequencing on Oxford Nanopore MinION/GridION typically results in read length N50s of 50kb with yields up to 10 Gb per flow cell. Megabase length reads are demonstrated using the Oxford Nanopore Rapid Sequencing Kit. In addition to DNA extraction, Nanobind can be used for tunable size selection up to 10 kb with >80% recovery of high MW DNA and >99% removal of short DNA. Gentle purification and high recovery ensure a greater number of long-reads >50 kb. This method enables library prep to be completed in less than 4 hours.