From Sampling to Sequencing: A Liquid Biopsy Pre-Analytic Workflow to Maximize Multi-Layer Genomic Information from a Single Tube.

Abstract

Simple SummaryLiquid biopsies seek to isolate tumor derived genetic material that circulates in blood plasma or cerebrospinal fluid. The less-invasive character of liquid biopsies combined with the option for serial analyses bears enormous potential for treatment monitoring or surveillance. We aimed to establish robust sampling protocols and pre-analytical workflows to allow for site independent multi-layer liquid biopsy testing. For an optimal usage of precious material, we explored sample stabilization in various conservation tubes and describe a protocol for the parallel isolation of cell-free DNA and RNA. Quantification and quality control steps were optimized for minimal sample use with both high sensitivity and reproducibility. We provide detailed step-by-step information on how to i) choose the best-suited protocol and ii) implement this in the liquid biopsy workflow. We believe that our study has potential to increase comparability of liquid biopsy approaches to bring these one step closer to routine clinical application.Liquid biopsies hold great promise for the management of cancer. Reliable liquid biopsy data depend on stable and reproducible pre-analytical protocols that comply with quality measures, irrespective of the sampling and processing site. We established a workflow for plasma preservation, followed by processing, cell-free nucleic acid isolation, quantification, and enrichment of potentially tumor-derived cell-free DNA and RNA. Employing the same input material for a direct comparison of different kits and protocols allowed us to formulate unbiased recommendations for sample collection, storage, and processing. The presented workflow integrates the stabilization in Norgen, PAX, or Streck tubes and subsequent parallel isolation of cell-free DNA and RNA with NucleoSnap and NucleoSpin. Qubit, Bioanalyzer, and TapeStation quantification and quality control steps were optimized for minimal sample use and high sensitivity and reproducibility. We show the efficiency of the proposed workflow by successful droplet digital PCR amplification of both cell-free DNA and RNA and by detection of tumor-specific alterations in low-coverage whole-genome sequencing and DNA methylation profiling of plasma-derived cell-free DNA. For the first time, we demonstrated successful parallel extraction of cell-free DNA and RNA from plasma samples. This workflow paves the road towards multi-layer genomic analysis from one single liquid biopsy sample.