Toward reliable biomarker signatures in the age of liquid biopsies - how to standardize the small RNA-Seq workflow.
Small RNA-Seq has emerged as a powerful tool in transcriptomics, gene expression profiling and biomarker discovery. Sequencing cell-free nucleic acids, particularly microRNA (miRNA), from liquid biopsies additionally provides exciting possibilities for molecular diagnostics, and might help establish disease-specific biomarker signatures. The complexity of the small RNA-Seq workflow, however, bears challenges and biases that researchers need to be aware of in order to generate high-quality data. Rigorous standardization and extensive validation are required to guarantee reliability, reproducibility and comparability of research findings. Hypotheses based on flawed experimental conditions can be inconsistent and even misleading. Comparable to the well-established MIQE guidelines for qPCR experiments, this work aims at establishing guidelines for experimental design and pre-analytical sample processing, standardization of library preparation and sequencing reactions, as well as facilitating data analysis. We highlight bottlenecks in small RNA-Seq experiments, point out the importance of stringent quality control and validation, and provide a primer for differential expression analysis and biomarker discovery. Following our recommendations will encourage better sequencing practice, increase experimental transparency and lead to more reproducible small RNA-Seq results. This will ultimately enhance the validity of biomarker signatures, and allow reliable and robust clinical predictions.
Department of Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Weihenstephaner Berg 3, 85354 Freising, Germany Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, Goethestraße 29, 80336 München, Germany.
Dominik
0000-0003-0460-6459
D
Buschmann
Department of Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Weihenstephaner Berg 3, 85354 Freising, Germany.
Anna
A
Haberberger
Department of Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Weihenstephaner Berg 3, 85354 Freising, Germany.
Benedikt
0000-0003-3878-0148
B
Kirchner
Department of Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Weihenstephaner Berg 3, 85354 Freising, Germany.
Melanie
M
Spornraft
Eurofins Medigenomix Forensik GmbH, Anzinger Straße 7a, 85560 Ebersberg, Germany Department of Anesthesiology, University Hospital, Ludwig-Maximilians-University Munich, Marchioninistraße 15, 81377 München, Germany.
Irmgard
0000-0002-8292-4148
I
Riedmaier
Department of Physiology, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Weihenstephaner Berg 3, 85354 Freising, Germany.
Gustav
G
Schelling
Department of Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Weihenstephaner Berg 3, 85354 Freising, Germany michael.pfaffl@wzw.tum.de.
Michael W
MW
Pfaffl
D015415:Biomarkers; D001706:Biopsy; D019295:Computational Biology; D020869:Gene Expression Profiling / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000302:isolation & purification; D057089:Pathology, Molecular / Q000379:methods
D015415:Biomarkers; D035683:MicroRNAs