Poster AGBT 2024
Broadening the input application of ResolveDNA genomic amplification to detection of SNV in circulating DNA
Jon S. Zawistowski, Swetha D. Velivela, Isai Salas-Gonzalez, Megan Justice, Tia A. Tate, Victor J. Weigman
BioSkryb Genomics, Durham, NC, USA
Uniform, allelically-balanced, and near-complete amplification of single-cell genomes has been enabled by ResolveDNA technology powered by primary template-directed amplification (PTA). This is achieved by attenuation of amplicon size, whereby the short amplicons have a low propensity to reamplify, manifesting with quasi-linear vs exponential amplification. Despite the power of this approach, this presents an inherent challenge for the amplification of cell-free and circulating tumor DNAs (cfDNA/ctDNA), which continue to hold promise as non-invasive diagnostic and monitoring tools for a multitude of disorders, due to their small size of only several hundred base pairs available as PTA input template. To provide a solution to the chief cf/ctDNA problem of template limitation, yet maintain the fidelity of PTA, we designed a scheme to concatenate short DNA fragments prior to amplification, increasing the size of input template to that which is amenable and optimal for PTA. This was undertaken using a size titration of synthetic DNA fragments as well as with cfDNA reference standards. Upon low-coverage sequencing, extending the template size of small fragments by ligation improved uniformity of exonic coverage as well as the number of loci detected within the cfDNA reference standard. Importantly, upon high-pass sequencing, while only 5/386 cfDNA reference standard loci were exposed with one detected single nucleotide variant (SNV) in the absence of our ligation strategy, 167 sites with 43 verified SNVs were detected post-ligation. Studies are underway to extend this proof of principle experiment to cfDNA isolated from plasma of a patient harboring a late-stage solid tumor with a defined missense mutational profile from a pan-cancer targeted panel, as well as to contrast the mutation detection sensitivity of existing methodology of cfDNA sequencing in the absence of amplification. In summary, the strategy presented here exposes single nucleotide variation from a realm of template previously not exposed with PTA and holds potential for assessment not only for non-invasive detection of tumor mutations but for a myriad of additional cfDNA applications including prenatal screening and heart disease.