Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
| Autor: | Jeffrey Houghton, Brian C. Haynes, Andrew G. Hadd, Gary J. Latham, Robert Zeigler |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
precision medicine General Chemical Engineering Biopsy Fine-Needle Biology FFPE Bioinformatics computer.software_genre drugs General Biochemistry Genetics and Molecular Biology DNA sequencing Issue 110 molecular diagnostics 03 medical and health sciences 0302 clinical medicine Software Targeted ngs Formaldehyde Neoplasms Humans cancer General Immunology and Microbiology business.industry General Neuroscience High-Throughput Nucleotide Sequencing bioinformatics Modular design Precision medicine Molecular diagnostics 030104 developmental biology Workflow FNA 030220 oncology & carcinogenesis Mutation oncology Next-generation sequencing Medicine Data mining business Multiplex Polymerase Chain Reaction Quality assurance computer |
| Zdroj: | Journal of Visualized Experiments : JoVE |
| ISSN: | 1940-087X |
| Popis: | All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench ("wet bench") and data analyses conducted using bioinformatics pipelines ("dry bench"). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible "tag" PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|