Impact of disruption methods on high molecular weight DNA extracted from archived frozen tissues

Abstract Background Tissue samples can provide high molecular weight (HMW) DNA for advanced genomic applications, such as long-read sequencing. Archived frozen tissues stored in biobanks or tissue repositories could retain DNA quality. However, the impact of different tissue disruption methods on DNA yield, purity and integrity remains incompletely characterized. Methods The impact of three disruption methods on DNA was initially compared using rabbit liver tissues: liquid nitrogen (LN) coarse smashing, LN fine grinding, and TissueLyser homogenization. Key parameters, including lysis time, homogenization buffer, agitation frequency, and running duration, were subsequently optimized to maximize DNA yield or preserve fragment integrity. Different disruption methods were further evaluated using archived frozen human liver, kidney, bladder, and pancreas tissues to assess their applicability across different tissue types. Results All methods yielded A260/280 ratios of ~ 1.8. LN fine grinding resulted in the lowest DNA yield, DNA integrity number (DIN) and A260/230. LN coarse smashing yielded optimal integrity (DIN > 8, peak size ~ 50 kb) but moderate yield, and prolonged lysis did not improve DNA recovery. The TissueLyser group with nuclease-free water had the highest yield and purity (A260/230 > 2.0) but the smallest fragment size (~ 25 kb). Buffer selection markedly affected the outcomes of TissueLyser homogenization. PBS achieved high integrity (DIN ~ 9, peak size ~ 60 kb), whereas ATL buffer (lysis solution of DNA extraction kit) produced smaller DNA fragments (~ 22 kb) but significantly increased the yield (1245.93 ± 168.37 ng/mg). Validation in human tissues demonstrated that the optimized TissueLyser protocol consistently outperformed the LN disruption methods across all four tissue types. Conclusions Tissue disruption should balance DNA recovery and integrity. TissueLyser homogenization offers a standardized and efficient approach for HMW DNA extraction. The buffer and parameters should be guided by the specific requirements of downstream applications.