Introduction and objectives Concern about the DNA quality for next-generation sequencing encourages use of dedicated preparative kits. The purpose of this study was to attempt to sequence ten stored DNA samples that had been prepared from human blood using phenol chloroform methods 12–17 years earlier, frozen at -70 C and not subjected to special treatments.

Methods The ten DNA samples that had been defrosted on multiple occasions, were defrosted again for library preparations using the Agilent SureSelect Target Enrichment System for Illumina paired-end multiplexed sequencing. Sequencing was performed on an Illumina HiSeq 2000 instrument for 2 × 100 base reads. Sequencing data were processed with RTA version 1.7.45 with default filter and quality settings, aligned to human genome build hg18 using CASAVA Eland pair algorithm, and demultiplexed with CASAVA 1.7.

Results All libraries passed stringent quality control steps at each step of library generation. More than 10 Giga bases (Gb) of sequence was generated from each read. High quality scores meant that even the data from the last of the 200 sequencing cycles were usable, with a cycle 200 median Phred score of 25. More than 3.3 million clusters passed filters for each read, and more than 86% of the sequence reads aligned to the human genome. For each sample, approximately 8 million primary sequence reads uniquely mapped to the captured region of interest.

Conclusions Extremely high quality DNA sequences can be obtained using stored DNA samples prepared many years earlier, and not subjected to any special treatments in the intervening years. The findings will be of particular importance to research communities where acquisition of new samples is not always possible.