It is common practice is to store extracted DNA at -20°C. We aimed to assess the influence of different numbers of freezing and thawing cycles (5, 10, 15, 20, and 25) on DNA preserved at this temperature. We extracted DNA from a Bacillus subtilis cell pellet using the QIAGEN Genomic-tip method and eluted the extracted DNA in water. This sample was sequenced and then diluted into either water or TE buffer. These stock solutions were individually aliquoted and frozen at -20°C. Aliquots were then thawed on ice before being re-frozen: a process which was repeated for a total number of 5, 10, 15, 20 or 25 cycles of thawing and re-freezing. The frozen/thawed samples were sequenced using Ligation Sequencing Kit. Prior to sequencing, the samples were SPRI size-selected.

Below, we present data on the effect of the freezing-thawing cycles on read N50. No impact on sequencing throughput was observed and all samples delivered 3-star performance: 8+ Gb in 48 h (on FLO-MIN106), equivalent to the Lambda DNA supplied with the Control Expansion pack (EXP-CTL001).

We recommend customers are aware of the effects of freezing and thawing samples multiple times, and advice to minimize these cycles in order to prevent reduction in observed read length.

Figure 1. The effect of freeze/thaw cycles on the observed read N50 of DNA sequencing libraries. DNA was extracted from B. subtilis and then stored in either TE or water. Subsequently, aliquots of extracted DNA were frozen at -20°C. These aliquots were then thawed on ice and re-frozen for different numbers of cycles and then prepared for sequencing using the Ligation Sequencing Kit. The prepared libraries were run on MinION and the read N50 (kb) and throughput (Gbases) noted. These data show that extracted DNA can start to degrade and deliver lower read N50s after several rounds of freezing and thawing.