A complete workflow for analysis of cfDNA: from plasma to variants

DATE: May 7, 2020

TIME: 7:00am PT

As cost of sequencing continues to drop, the throughput and complexity of NGS assays have risen precipitously. At the same time, the types of samples being used for these assays have expanded as researchers find value in studying low input, degraded biological samples, such as cell free DNA (cfDNA). Obtaining actionable information from clinical samples is challenging due to loss during extraction, low conversion during NGS library preparation, drop in complexity during target enrichment, and sequencing and PCR errors that lead to low limits of detection. Here, we present a complete workflow for effective analysis of <1% AF variants in cfDNA. Our magnetic bead-based extraction kit provides complex, high yield recovery of cfDNA. A novel library prep chemistry delivers higher complexity and coverage than conventional TA ligation-based workflows, enabling highly sensitive low-frequency variant detection. In addition, fixed unique molecular index (UMI) sequences aid strand-specific molecular indexing by independently tagging the top and bottom strands, and can be used for various deduplication and error correction strategies. Our target enrichment strategies maintain high library diversity and on-target rates to enable low frequency variant calling regardless of panel size. We pair these chemistries with modularly designed automation protocols which provide users with safe stopping points, enabling customizable, high throughput solutions. By combining a high conversion extraction and library preparation chemistry with efficient hybridization capture, we demonstrate an effective approach to extract information from low quantity challenging samples.

Learning Objectives:

• Describe how to increase cfDNA extraction efficiency using magnetic bead–based technology.
• Demonstrate how to maximize conversion, coverage and complexity from low inputs of cfDNA during library preparation.
• Utilize fixed UMIs for bioinformatic error correction to reduce sequencing and PCR errors.
• Discuss how to increase throughput, reduce hands-on time, and minimize errors during complex NGS assays.

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LabRoots is approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E. ® Program. By attending this webinar, you can earn 1 Continuing Education credit once you have viewed the webinar in its entirety.  CE credits are available for this webinar up to 2 years from the date of the live broadcast.