Abstract 496: Novel sample isolation and immortalization method for DNA and RNA to extend translational research capabilities
Authors: Spinosa John Christopher , Young Brandon , Aguilar Vincent , Parikh Baiju 
Published: June 2022

Abstract
Background: Translational cancer research is dependent on well annotated human samples, but scarcity of specimen DNA and RNA hinders research and novel diagnostic testing. We demonstrate a novel nucleic acid isolation and anchoring approach that facilitates uniform, consistent replication of the isolated and captured DNA and RNA. This approach allows for expanded investigations of precious samples and increases the value of biobanked material.
Methods: Our simplSEQ replication methodology enzymatically adds homopolymeric tails to the 3’ end of DNA and RNA, with a terminal dideoxy biotin-modified NTP, to attach the nucleic acids to streptavidin beads. The 3’ immobilized nucleic acids are copied using complementary homopolymers. The simplSEQ advantage is that molecules of all sizes are attached and copied from their 3’ ends yielding cDNA copies that are full-length representations of the input. Various DNA samples were investigated to determine the characteristics of cDNA copies as well as stability of bound nucleic acids. Samples evaluated included Human 1,000 genome project, bacterial genomes, commercial DNA controls, and synthesized DNA molecules. The samples represent a range of DNA sizes, AT/GC content and Variant Allele Frequencies (VAF). NGS assays quantified the presence of full-length genomic sequences. Variant analysis determined accuracy of copies based on comparison with published data (NA12878). Synthetic DNA oligonucleotides representing regions in the human genome and constructed to enable molecular counting independent of adapter ligation efficiency. All sample types were replicated, sequenced, and analyzed for coverage and uniformity. This replication process was repeated multiple times.
Results: Three replicates of each DNA type were attached to streptavidin beads and subjected to multiple rounds of the simplSEQ protocol. Analysis of overall coverage and variant calling for human NA12878 replicates compared to the platinum standard NA12878 genome show an average coverage ranging from 27x-32x with &gt;99% correlation between replicates. Sensitivity ranging from 98.6%-99.5%, with a precision range from 96.0% to 98.0% for 6 replicates. Bacterial samples with coverage over 100x and &gt;99% correlation. Synthetic DNA oligonucleotide pools with coverage over 10,000x and &gt;99% correlation.
Conclusion: Our novel enzymatic tailing methodology produces highly accurate copies of full-length nucleic acids. This technology obviates sample scarcity for genomic evaluations and allows for expanded hypothesis testing in translational research. This methodology also provides an option for extended storage of nucleic acids from which replicates can be repeatedly generated to allow for reinterrogation.
Citation Format: John Christopher Spinosa, Brandon Young, Vincent Aguilar, Baiju Parikh. Novel sample isolation and immortalization method for DNA and RNA to extend translational research capabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 496.