RNA sequencing (RNA-seq) has become an indispensable tool to identify disease associated transcriptional profiles and determine the molecular underpinnings of diseases. However, the broad adaptation of the methodology into the clinic is still hampered by inconsistent results from different RNA-seq protocols and involves further evaluation of its analytical reliability using patient samples. Here, researchers at the University of Helsinki applied two commonly used RNA-seq library preparation protocols to samples from acute leukemia patients to understand how poly-A-tailed mRNA selection (PA) and ribo-depletion (RD) based RNA-seq library preparation protocols affect gene fusion detection, variant calling, and gene expression profiling.
Overall, the protocols produced similar results with consistent outcomes. Nevertheless, the PA protocol was more efficient in quantifying expression of leukemia marker genes and showed better performance in the expression-based classification of leukemia. Independent qRT-PCR experiments verified that the PA protocol better represented total RNA compared to the RD protocol. In contrast, the RD protocol detected a higher number of non-coding RNA features and had better alignment efficiency. The RD protocol also recovered more known fusion-gene events, although variability was seen in fusion gene predictions.
Experimental design and data analysis workflow of the study
a Bone marrow aspirates were collected from two ALL and two AML patient samples. Total RNA was isolated and used for RNA-seq library preparation by RD and PA protocols from the same RNA source. Altogether, two PA enriched and six RD libraries were constructed. The ALL 542 and AML 800 samples were used to analyze variation between library construction protocols and ALL 668 and AML 1867 samples used for technical variation comparison. b The flow chart shows the steps and tools used for RNA-seq data analysis. c Clinical characteristics of the leukemia patients
The overall findings provide a framework for the use of RNA-seq in a precision medicine setting with limited number of samples and suggest that selection of the library preparation protocol should be based on the objectives of the analysis.