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Complete Workflow for Automated cell-free DNA Extraction and Somatic Mutation Detection
EP24892
Poster Title: Complete Workflow for Automated cell-free DNA Extraction and Somatic Mutation Detection
Submitted on 27 Nov 2016
Author(s): Tatiana Ivanova, Amanda Fan, Alex Yeo, Elian Rakhmanaliev, Pramila Ariyaratne, Gerd Michel
Affiliations: Vela Research Pte. Ltd., Singapore
This poster was presented at AMP 2016 Annual Meeting, Nov. 10-12, 2016, Charlotte, North Carolina, USA
Poster Views: 2,608
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Poster Information
Abstract: Circulating cell-free DNA (cfDNA) has emerged as an important biomarker in cancer diagnostics and non-invasive progression monitoring of various clinical conditions. This has resulted in the development of new in vitro diagnostics (IVD) cfDNA assays. Challenges encountered in these developments related to the efficient extraction of cfDNA from liquid biopsies, often yielding low quantities of highly fragmented DNA. As assays for cfDNA are typically intended to identify genetic variants present at very low allelic frequencies, many of the established detection technologies are driven to the edge of their performance.
We developed a magnetic bead-based cfDNA extraction kit Sentosa SX cfDNA Kit (4x8) and optimized it for use on the Vela Sentosa SX101 platform. Sentosa SX101 is a CE-IVD certified robotic liquid handling system for nucleic acid extraction, PCR set-up and Next-Generation Sequencing (NGS) library preparation. We compared performance of the Sentosa SX cfDNA Kit (4x8) with a column-based cfDNA extraction kit. Integrity of cfDNA extracted by both methods was assessed using ALU repeats qPCR assay. Quality of the extracted cfDNA was tested using an NGS-based Sentosa SQ CRC Panel (4x8).
The Sentosa SX cfDNA Kit (4x8) utilizes 4 mL of human plasma as sample input and can process up to eight samples per run. In this pilot study DNA was extracted from plasma samples with 3.0, 1.5 and 0.75 ng spiked-in fragmented HCT116 gDNA (KRAS G13D positive) using Sentosa SX cfDNA and column-based cfDNA extraction kits, respectively. Fragment size of HCT116 gDNA was ~170 bp (confirmed by Bioanalyzer). The ALU247/115 ratio for DNA extracted by the Sentosa SX cfDNA kit was 0.19-0.28 and for the column-based extraction method >0.7 (expected ratio for cfDNA is less than 0.5 and for gDNA is 1.0). Amount and quality of DNA for all samples extracted by both methods was sufficient to prepare NGS libraries using Sentosa SQ CRC Panel (4x8). KRAS G13D mutation was detected in all samples extracted by the Sentosa SX cfDNA kit and no KRAS G13D mutation was detected in any of column-based extracted samples.
The Sentosa SX cfDNA Kit (4x8) selectively extracts cfDNA over high molecular weight gDNA. The Sentosa SX cfDNA Kit (4x8) appears as an efficient and reliable solution for cfDNA extraction from human plasma samples. Integration into the Sentosa qPCR- and NGS-based workflows makes the Sentosa SX cfDNA Kit (4x8) a universal in vitro diagnostics tool, which can be used in combination with various IVD assays.
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