Paired DNA and RNA sequencing is an increasingly popular method used to produce libraries from FFPE samples.
Whilst traditional sequencing methods can present challenges distinguishing between the two types of nucleic acids, thus presenting potential biases in amplification, a total prep workflow streamlines sequencing by processing DNA and RNA from one sample in a single tube, maximizing efficiency.
This poster presents a solution that can offer results in less than 1.5 days, producing sequencing-ready libraries from total nucleic acid samples.
Download this poster to learn more about a method that can:
- Ensure robust quality control
- Efficiently segregate RNA and DNA reads within the input data for downstream analysis
- Demonstrate comparable performance from high- and low-quality FFPE input material
The KAPA Total Prep workflow facilitates simultaneous sequencing of both DNA and RNA from a single sample within a single tube, optimizing time and resource utilization. The workflow takes under 1.5 days to generate sequencing-ready, target-enriched libraries from samples containing total nucleic acid as initial input. LIBRARY QUALITY ASSESSMENT Libraries produced using low-quality Formalin-Fixed Paraffin-Embedded (FFPE) input material exhibit comparable sequencing quality compared to those from high-quality FFPE input material. DNA DATA EVALUATION DNA data derived from both low-quality and high-quality FFPE input material demonstrate comparable performance in germline variant calling. DNA DATA EVALUATION (Continued) DNA data obtained from somatic variant reference samples show reliable performance in somatic variant calling. The data represent the mean of three replicates for each sample, processed using the KAPA Total Prep FFPE workflow and enriched with the KAPA HyperCap Oncology panel. Each library was subsampled to 10 million read pairs before undergoing processing using the KAPA Total Prep Bioinformatics pipeline. Detected variants were compared to the NA24385 benchmark variants within the target regions of the KAPA HyperCap Oncology panel. Expression patterns of variant-associated genes altered in reference samples. Benchmark variants within the target regions of the KAPA HyperCap Oncology panel were used for the evaluation. We implemented a bioinformatics pipeline tailored specifically for the KAPA Total Prep workflow, streamlining data processing and ensuring robust quality control. The pipeline efficiently segregates RNA and DNA reads within the input data, enabling focused downstream analysis targeting distinct molecular types. *Note: GRCh38 reference files are provided as default in the available package of the KAPA Total Prep Bioinformatics pipeline. KAPA Total Prep Bioinformatics pipeline KAPA Total Prep FFPE workflow RNA DATA INTEGRATION RNA data generated from reference samples show steady performance in detection of fusion genes and alternative splicing events. DNA RNA High-quality FFPE Compromised FFPE High-quality FFPE Compromised FFPE Duplication rate after UMI processing 1.80% 3.40% 2.30% 8.40% Genome mapping rate 100% 100% 99.40% 99.30% Median coverage depth 998 X 691 X 45 X 76 X On-target rate 86.00% 88.50% 96.70% 98.50% Error rate 0.47%% 0.57% NA NA Artifact rate 0.07% 0.04% NA NA Fraction of targets with at least 30X coverage depth 99.20% 99.20% NA NA Fold-80 base penalty 2.90 2.80 NA NA Transcript mapping rate NA
