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

Cancers

卷:13

Pitithat Puranachot¹  Natalie Stumpf²  Stefan M. Pfister²  Agnes M. E. Finster²  Tatjana Wedig²  Nathalie Schwarz²  Kristian W. Pajtler²  Fabian Rosing²  Paulina S. Schad²  Kendra K. Maass² 

[1] Division of Applied Bioinformatics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany;

[2] German Cancer Research Center (DKFZ), Division of Pediatric Neurooncology, Hopp-Children’s Cancer Center Heidelberg (KiTZ) and German Cancer Consortium (DKTK), 69120 Heidelberg, Germany;

关键词: liquid biopsy;    cell-free DNA;    cell-free RNA;    blood preservation tubes;    CSF;    size selection;   

DOI  :  10.3390/cancers13123002

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown