This presentation introduces a novel single-tube strategy for combined RNA and DNA library preparation for Next Generation Sequencing (NGS). This approach aims to address the inherent variability and practical challenges associated with traditional separate DNA and RNA assays from distinct samples. The single-tube workflow simultaneously converts both DNA and RNA from a single biological sample into sequencing libraries, utilizing a tagging mechanism to discriminate between DNA and RNA reads during downstream analysis. Key advantages of this method include significantly reduced turnaround and hands-on time, conservation of precious and limited samples (such as FFPE tumor biopsies), and the preservation of native DNA:RNA ratios by enabling total nucleic acid extraction without enzymatic degradation steps. Crucially, it mitigates bias arising from tumor heterogeneity, providing more reliable and comprehensive paired genomic and transcriptomic profiling. This integrated approach facilitates orthogonal verification of DNA variant calls with RNA data and enhances the detection and functional interpretation of complex splicing variants, which are vital for understanding disease mechanisms, identifying candidate driver mutations, and advancing targeted therapies in fields like oncology and hereditary cancer research.
Exploring a Single-Tube Workflow for Combined RNA and DNA Library Preparation
0:00 Mariana K introduces a "single tube strategy" for combined RNA and DNA library preparation for Next Generation Sequencing (NGS), emphasizing its advantages for understanding disease and phenotypic variation.
0:17 Traditional methods often analyze DNA and RNA separately from distinct cell populations, increasing sample variability and being cumbersome and impractical.
1:04 The "single tube workflow" streamlines the process by simultaneously generating DNA and RNA libraries from a single sample in the same tube. RNA molecules are tagged to allow discrimination of DNA and RNA reads during data analysis.
1:41 Target enrichment can be performed simultaneously for both DNA and RNA, capturing specific regions of interest and their corresponding transcripts. This diminishes sequencing costs, enables investigation of rare variants, and eliminates the need for ribosomal RNA depletion.
2:10Key Takeaway: Time & Efficiency. The single-tube workflow significantly reduces turnaround time (by about 1.5 hours compared to traditional sequential methods) and requires less hands-on time, potentially needing only one technician instead of two for parallel preps.
3:24Key Takeaway: Sample Conservation. This method conserves precious samples, such as FFPE tumor tissues with limited availability, as total nucleic acid is used, allowing both RNA and DNA data from the same tissue section.
3:52 Using total nucleic acid extraction (an upfront step) avoids the need for RNase or DNase treatments, reducing loss of DNA/RNA and preserving the original DNA:RNA ratios from the sample (e.g., 1:1 to 1:5 for FFPE tumors, 1:1 to 1:2 for fresh-frozen tumors).
4:49Key Takeaway: Reduced Variability & Improved Profiling. The single-tube strategy reduces bias from tumor heterogeneity (differences within cancer cells of a single tumor) by using the same sample, enabling more reliable paired DNA and RNA tumor profiling.
5:50 This approach provides paired DNA and RNA profiling from the same biological sample, leading to a deeper understanding of tumor behavior. DNA reads detect biomarkers (SNVs, indels, CNVs), while RNA reads detect gene fusions, exon skipping, splicing isoforms, and gene expression levels.
6:29 RNA sequencing data provides orthogonal verification of DNA variant calls, identifying expressed variants that are more likely associated with biological effects in cancer (e.g., approximately one-third of somatic SNVs in coding regions are observable in RNA). This acts as a biological filter for candidate driver mutations.
7:16 RNA sequencing also aids in classifying DNA variants in hereditary cancer, particularly for challenging splicing variants located far from coding sequences. Splicing profiling by RNA sequencing improves detection and provides functional evidence for accurate interpretation of putative splicing variants.
8:17Overall Summary: The single-tube DNA and RNA sequencing strategy uncovers molecular mechanisms, saves time and resources, preserves precious samples, and reduces bias from sample variability, ultimately enabling more reliable tumor profiling and genotype-phenotype correlations.