Review Article | Published:

Translating RNA sequencing into clinical diagnostics: opportunities and challenges

Nature Reviews Genetics volume 17, pages 257271 (2016) | Download Citation


With the emergence of RNA sequencing (RNA-seq) technologies, RNA-based biomolecules hold expanded promise for their diagnostic, prognostic and therapeutic applicability in various diseases, including cancers and infectious diseases. Detection of gene fusions and differential expression of known disease-causing transcripts by RNA-seq represent some of the most immediate opportunities. However, it is the diversity of RNA species detected through RNA-seq that holds new promise for the multi-faceted clinical applicability of RNA-based measures, including the potential of extracellular RNAs as non-invasive diagnostic indicators of disease. Ongoing efforts towards the establishment of benchmark standards, assay optimization for clinical conditions and demonstration of assay reproducibility are required to expand the clinical utility of RNA-seq.

Key points

  • RNA-based measurements have the potential for application across diverse areas of human health, including disease diagnosis, prognosis and therapeutic selection. Current clinical applications include infectious diseases, cancer, transplant medicine and fetal monitoring.

  • RNA sequencing (RNA-seq) allows for the detection of a wide variety of RNA species, including mRNA, non-coding RNA, pathogen RNA, chimeric gene fusions, transcript isoforms and splice variants, and provides the capability to quantify known, pre-defined RNA species and rare RNA transcript variants within a sample. In addition to differential expression and detection of novel transcripts, RNA-seq also supports the detection of mutations and germline variation for hundreds to thousands of expressed genetic variants, facilitating assessment of allele-specific expression of these variants.

  • Circulating RNAs and small regulatory RNAs, such as microRNAs, are very stable. These RNA species are vigorously being tested for their potential as biomarkers. However, there are currently few agreed upon methods for isolation or quantitative measurements and a current lack of quality controls that can be used to test platform accuracy and sample preparation quality.

  • Analytical, bioinformatic and regulatory challenges exist, and ongoing efforts toward the establishment of benchmark standards, assay optimization for clinical conditions and demonstration of assay reproducibility are required to expand the clinical utility of RNA-seq.

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The authors acknowledge funding from the Ben and Catherine Ivy Foundation and the National Center for Advancing Translational Sciences (exRNA Signatures Predict Outcomes After Brain Injury; UH3-TR000891). Research was also supported by a Stand Up To Cancer – Melanoma Research Alliance Melanoma Dream Team Translational Cancer Research Grant. Stand Up To Cancer is a programme of the Entertainment Industry Foundation administered by the American Association for Cancer Research. The authors apologize to those whose work could not be cited or discussed owing to space constraints.

Author information


  1. Center for Translational Innovation, Translational Genomics Research Institute, Phoenix, Arizona 85004, USA.

    • Sara A. Byron
  2. Neurogenomics Division, Translational Genomics Research Institute, Phoenix, Arizona 85004, USA.

    • Kendall R. Van Keuren-Jensen
    •  & David W. Craig
  3. Pathogen Genomics Division, Translational Genomics Research Institute, Flagstaff, Arizona 86001, USA.

    • David M. Engelthaler
  4. Integrated Cancer Genomics Division, Translational Genomics Research Institute, Phoenix, Arizona 85004, USA.

    • John D. Carpten


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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to David W. Craig.


Next-generation sequencing

(NGS). High-throughput, massively parallel sequencing technology that is used in various applications, including whole-genome sequencing, exome sequencing and RNA sequencing.

Open platform

A technology platform that does not depend on genome annotation, or on predesigned species-specific or transcript-specific probes, for transcript measurement. RNA-seq technology functions as an open platform allowing for unbiased detection of both known and novel transcripts.

Single nucleotide variants

(SNVs). Single nucleotide (A, T, G or C) alterations in a DNA sequence.

Reference standards

Highly characterized and standardized control materials that are used to ensure accuracy and comparability of assays.

Spearman correlation

Statistical measure of the strength of association between two rank-ordered variables.


Evaluation of closely situated mutations to determine whether they reside on the same or different alleles.

Break-apart probes

A DNA probe system used to detect rearrangements involving specific loci. Probes for a region 5′ of the designated breakpoint are labelled with one colour and probes for a region 3′ of the breakpoint are labelled with another colour. An overlapping signal (such as yellow for red and green probes) indicates a normal pattern, whereas distinct signals (that is, red and green) indicate the presence of a rearrangement.

Structural variants

Genomic variants, other than single-nucleotide variants, involving large regions of DNA, including insertions, deletions, inversions and duplications.

Single nucleotide polymorphisms

(SNPs). Single nucleotide alterations that represent single base-pair variation at a specific DNA position among individuals, the majority of which are inherited.

Nonsense-mediated decay

A translation-coupled RNA decay mechanism whereby aberrant mRNAs with premature stop codons are recognized and degraded.

Expression quantitative trait loci

(eQTLs). Genomic loci that regulate the quantitative phenotypic trait of gene expression. Genetic markers at these loci are associated with measurable changes in gene expression.

RNA split reads

RNA sequencing reads that are split — for example, to accommodate exon junctions.

Extracellular RNAs

(exRNAs). RNAs found outside of the cell, they can be protected within vesicles or in association with RNA-binding proteins, and they can include exogenous sequences.

Clinical Laboratory Improvement Amendments

(CLIA). All laboratory testing on humans in the United States is regulated by The Centers for Medicare and Medicaid Services through CLIA. The purpose is to ensure quality and uniformity of laboratory tests.

Metagenomic RNA-seq

A method of sequencing the entirety of the available RNA in a complex (for example, clinical or environmental) sample, which may or may not include steps to subtract the host RNA to improve or enrich for microbial RNA.

RNA-based amplicon sequencing

A method of direct sequencing of cDNA amplicons of RNA targets from a clinical sample. This can be multiplexed and can involve RNA viral genomes, microbial or host mRNA transcripts, or exogenous RNA targets.


The totality of the genomic content of microbial community members in a complex (for example, clinical or environmental) sample. In the human microbiome, each body site has its own unique microbiome; the entirety of the microbiome on and in an individual person is considered that person's pan-microbiome.

Companion diagnostic

In vitro diagnostic tests that provide information critical for the safe and effective use of a corresponding therapeutic agent. These tests are used to select patients for treatment with specific agents, including identifying patient populations with predicted efficacy as well as those that should not receive the agent due to a low likelihood of effectiveness or possible serious adverse events from the therapy.

Variant call format

Standard text file format for storing genomic sequence variant data, with each line of the file describing a variant present at a specific genomic region or position.

Binary alignment/map format

(BAM format). Standard file format for storing sequencing reads with alignments. BAM files are binary representations of the sequence alignment/map (SAM) format.

In vitro diagnostic tests

Laboratory tests used to detect health conditions, infections or diseases. These diagnostic tests are performed using a sample collected from the patient without direct physical interaction between the test and the patient. In the United States, in vitro diagnostics are regulated by the US Food and Drug Administration (FDA).

Triple-negative breast cancer

A breast cancer subtype characterized as oestrogen receptor-negative, progesterone receptor-negative and ERBB2 (also known as HER2)-negative.

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