ABSTRACT
RNA sequencing (RNAseq) technology has become increasingly important in precision medicine and clinical diagnostics, and emerged as a powerful tool for identifying protein-coding genes, performing differential gene analysis, and inferring immune cell composition. Human peripheral blood samples are widely used for RNAseq, providing valuable insights into individual biomolecular information. Blood samples can be classified as whole blood (WB), plasma, serum, and remaining sediment samples, including plasma-free blood (PFB) and serum-free blood (SFB) samples that are generally considered less useful byproducts during the processes of plasma and serum separation, respectively. However, the feasibility of using PFB and SFB samples for transcriptome analysis remains unclear. In this study, we aimed to assess the suitability of employing PFB or SFB samples as an alternative RNA source in transcriptomic analysis. We performed a comparative analysis of WB, PFB, and SFB samples for different applications. Our results revealed that PFB samples exhibit greater similarity to WB samples than SFB samples in terms of protein-coding gene expression patterns, detection of differentially expressed genes, and immunological characterizations, suggesting that PFB can serve as a viable alternative to WB for transcriptomic analysis. Our study contributes to the optimization of blood sample utilization and the advancement of precision medicine research. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00121-1.
ABSTRACT
Accurate indel calling plays an important role in precision medicine. A benchmarking indel set is essential for thoroughly evaluating the indel calling performance of bioinformatics pipelines. A reference sample with a set of known-positive variants was developed in the FDA-led Sequencing Quality Control Phase 2 (SEQC2) project, but the known indels in the known-positive set were limited. This project sought to provide an enriched set of known indels that would be more translationally relevant by focusing on additional cancer related regions. A thorough manual review process completed by 42 reviewers, two advisors, and a judging panel of three researchers significantly enriched the known indel set by an additional 516 indels. The extended benchmarking indel set has a large range of variant allele frequencies (VAFs), with 87% of them having a VAF below 20% in reference Sample A. The reference Sample A and the indel set can be used for comprehensive benchmarking of indel calling across a wider range of VAF values in the lower range. Indel length was also variable, but the majority were under 10 base pairs (bps). Most of the indels were within coding regions, with the remainder in the gene regulatory regions. Although high confidence can be derived from the robust study design and meticulous human review, this extensive indel set has not undergone orthogonal validation. The extended benchmarking indel set, along with the indels in the previously published known-positive set, was the truth set used to benchmark indel calling pipelines in a community challenge hosted on the precisionFDA platform. This benchmarking indel set and reference samples can be utilized for a comprehensive evaluation of indel calling pipelines. Additionally, the insights and solutions obtained during the manual review process can aid in improving the performance of these pipelines.