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Comparison of cfDNA Reference Material Prepared using Enzymatic Fragmentation or Sonication for the Validation of Liquid Bi
Comparison of cfDNA Reference Material Prepared using Enzymatic Fragmentation or Sonication for the Validation of Liquid Bi
Submitted on 08 Nov 2018

Hannah Child, Aldo Mele, Katarzyna Wilczynska, Julie Wickenden
Horizon Discovery Group
This poster was presented at AMP Annual Meeting 2018
Poster Views: 311
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Poster Abstract
The genotype of multiple actionable loci can now be determined from as little as 10ng DNA extracted from a routine patient blood sample, helping to direct therapeutic decision making and improve overall clinical outcome faster for the patient. However, in order to realise the full potential of this emerging technology, sequencing labs
need to ensure accuracy by validating a range of challenging new techniques. This includes the ability to extract cfDNA from blood samples, sequence it at new levels of sensitivity (down to 0.1% limit of detection) and establish effective bioinformatics pipelines. Reference materials that closely mimic real cfDNA samples are critical to support this effort. This study investigates the use of sonicated or enzymatically sheared cell-line derived DNA as alternative methods to create the most commutable cfDNA reference material for the validation of liquid biopsy assays. Methods: DNA was extracted from well-characterised cancer cell lines and fragmented to 160bp using either sonication or enzymatic shearing, as assessed by Tapestation. In addition, a size selection step was included for investigation into the ability to purity the fragment peak to a size distribution profile most similar to real cfDNA samples. Test samples were validated for the presence of 8 onco-relevant mutations by ddPCR, allowing for accurate variant allele frequency to be determined. The sample set was tested by NGS on Thermo breast cfDNA assay, and Illumina TST-15 assay, following the manufacturer’s instructions. Results: Tapestation analysis confirmed that both sonication and enzymatic shearing
produced cfDNA with an average fragment size of 160-170bp. A size selection step proved useful to
concentrate the amount of DNA within the desired fragment size range. Variant detection by ddPCR
confirmed the presence of 8 mutations across 4 genes (EGFR, KRAS, NRAS and PIK3CA) at either 0.1%
or 5% variant allele frequency. Analysis by NGS confirmed that all variants were detectable at the correct allele frequency as expected. Conclusion: Results demonstrate that both sonication and enzymatic shearing can be used to create cfDNA reference material with an average fragment size of 160-170bp to closely mimic cfDNA from real patient samples. This study has allowed a new manufacturing method to be investigated,utilising enzymatic shearing and a size selection step in order to further improve the commutability of high quality cell-line derived cfDNA reference material for the validation of liquid biopsy assays.Report abuse »
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