A comprehensive model of DNA fragmentation for the preservation of High Molecular Weight DNA

Abstract

ABSTRACTFor long-read sequencing applications, shearing of DNA is a significant issue as it limits the read-lengths generated by sequencing. During extraction and storage of DNA the DNA polymers are susceptible to physical and chemical shearing. In particular, the mechanisms of physical shearing are poorly understood in most laboratories as they are of little relevance to commonly used short-read sequencing technologies. This study draws upon lessons learned in a diverse set of research fields to create a comprehensive theoretical framework for obtaining high molecular weight DNA (HMW-DNA) to support improved quality management in laboratories and biobanks for long-read sequencing applications.Under common laboratory conditions physical and chemical shearing yields DNA fragments of 5-35 kilobases (kb) in length. This fragment length is sufficient for DNA sequencing using short-read technologies but for Nanopore sequencing, linked reads and single molecular real time sequencing (SMRT) poorly preserved DNA will limit the length of the reads generated.The shearing process can be divided into physical and chemical shearing which generates different patterns of fragmentation. Exposure to physical shearing creates a characteristic fragment length where the main cause of shearing is shear stress induced by turbulence. The characteristic fragment length is several thousand base pairs longer than the reads produced by short-read sequencing as the shear stress imposed on short DNA fragments is insufficient to shear the DNA. This characteristic length can be measured using gel electrophoresis or instruments for DNA fragment analysis,. Chemical shearing generates randomly distributed fragment lengths visible as a smear of DNA below the peak fragment length. By measuring the peak of the DNA fragment length distribution and the proportion of very short DNA fragments, both sources of shearing can be measured using commonly used laboratory techniques, providing a suitable quantification of DNA integrity of DNA for sequencing with long-read technologies.