Recently, various test methods using NGS technology (Panel Sequencing, WES, and WGS) are being utilized to diagnose rare genetic diseases. The test method is generally selected at the discretion of the physician upon evaluation of the patient's symptoms. In the past, CMA (Chromosomal Microarray Analysis) and Sanger Sequencing have been recommended as first-tier tests. But with the decrease in the cost of NGS analysis and accumulation of genetic knowledge through research, WES/WGS testing is being recommended as a first-tier test for more and more cases. However, WES is not the best method for all patients with rare diseases. In this post, I will introduce when and why WES testing is recommended as a first-tier test based on recent studies.

What is a first-tier test? It is the first diagnostic test that is conducted when an undiagnosed patient visits a hospital.

Infant patients with suspected rare diseases

The effectiveness of WES was analyzed in comparison to conventional testing methods on infants with suspected rare diseases under 2 years of age recruited between 2014 and 2015 at the Royal Children's Hospital in Melbourne.1

In the study, WES testing showed a higher diagnosis rate than standard tests including single gene sequencing, targeted gene panel, methylation study, and mitochondrial mutation panel. Based on these results, WES is recommended as first-tier testing for children for the following reasons.

  • Children are often undiagnosed due to lack of information necessary for diagnosis; the symptoms are incomplete and it is before the onset of other symptoms. Therefore, in such cases, examining the entire exome at once via WES may speed up diagnosis.
  • Prompt response through early diagnosis can prevent or delay the onset of serious symptoms.
  • Furthermore, if the patient's relatives are of reproductive age, diagnosis can have a significant impact on family planning.

For example, the diagnosis of Cohen syndrome is often delayed because symptoms usually appear in late childhood or adolescence. The complexity and severity of symptoms are often high in most early-onset diseases, for which a delay in diagnosis can be life-threatening. Therefore, it can be especially helpful for pediatric patients to receive an early diagnosis through WES.

Group of diseases with new gene-disease findings

Despite active research on rare diseases, there are still many diseases with unknown causes. Around 300 new pathogenic variants are discovered every year, and there are many studies that show that more information was revealed for symptoms that are more clearly defined, that occur throughout the body, or that are severe.

In this study2 published in Clinical Reviews in Allergy & Immunology last March, results of NGS diagnostic studies in patients with primary immunodeficiencies were analyzed. From the findings3 presented by the International Union of Immunological Societies (IUIS) in 2020, PID is a disease group for which new information is rapidly accumulating, and as a result of comparison with other test methods, WES testing showed a higher diagnosis rate.

WES can include up-to-date information in the analysis and prevent repeated testing because a new diagnosis can be made through data reanalysis with just one test. For these reasons, it is advantageous to use WES as a first-tier test in a disease group where many new disease-related genes are revealed.

ASD is the most common neurodevelopmental disorder (NDD), affecting 1 in 160 people worldwide and is an extremely heterogeneous group of disorders. Even patients with the most common monogenic ASD from the FMR1 gene mutation are known to account for only 2-4% of all patients. In addition, ASD patients often show a wide variety of symptoms.

In this study4, CMA and FMR1 testing, which have been recommended as first-tier tests for ASD, were compared to WES testing. 75% of patients were diagnosed by WES, 20.4% by CMA, and 4.5% by FMR1 testing.
Based on this high diagnostic rate, the researchers recommend WES as a first-tier test for ASD patients if Fragile X Syndrome is not suspected.

WES is also more beneficial than CMA in terms of cost and TAT (turnaround time). In terms of technology, SNVs (Single Nucleotide Variants) can be detected with WES, and CNVs (Copy Number Variants) can be accurately detected with the development of bioinformatics technology.

Similarly, in the systematic review paper5, 30 studies related to CMA and WES tests for NDD disease were analyzed, and results showed a high diagnosis rate by WES. The researchers propose that WES be the first-tier test for NDD diagnosis and that CMA be performed when undiagnosed.

With the improvement of WES technology, it is recommended to use WES as a first-tier test instead in disease groups where CMA was traditionally recommended as a first-tier test.


We looked into the background on why WES is recommended as a first-tier test through research studies on its effectiveness in rare disease diagnosis. ACMG, the most renowned organization in genetics and genomics, strongly recommends WES/WGS testing for pediatric patients with congenital anomalies or intellectual disability.6,7
And as research studies on various patient cohorts accumulate, the recommendation of WES/WGS as primary or secondary testing may be expanded to additional disease groups.

Various NGS-based rare disease screening methods are increasingly being used in medicine. However, each test has its strengths and weaknesses, as well as limitations. It is important for patients and clinicians to have the right information in order to determine the most appropriate test method.

If you have any questions or if there is information you need in the process of choosing the right rare disease diagnostic method, please feel free to reach out to us. We will continue to provide useful content for both patients and clinicians.


  1. Stark, Z. et al. A prospective evaluation of whole-exome sequencing as a first-tier molecular test in infants with suspected monogenic disorders. Genet Med 18, 1090–1096 (2016).
  2. Vorsteveld, E.E. et al. Next-Generation Sequencing in the Field of Primary Immunodeficiencies: Current Yield, Challenges, and Future Perspectives. Clinic Rev Allerg Immunol 61, 212–225 (2021).
  3. Tangye, S. al. Human inborn errors of immunity: 2019 update on the classification from the International Union of Immunological Societies Expert Committee. Journal of Clinical Immunology, 40(1), 24–64 (2020).
  4. Arteche-López APa Towards a Change in the Diagnostic Algorithm of Autism Spectrum Disorders: Evidence Supporting Whole Exome Sequencing as a First-Tier Test. Genes, 12(4), 560. (2021).
  5. Srivastava S, et al. Meta-analysis and multidisciplinary consensus statement: exome sequencing is a first-tier clinical diagnostic test for individuals with neurodevelopmental disorders. Genet Med, 2413-2421 (2019)
  6. Manickam, K. et al. Exome and genome sequencing for pediatric patients with congenital anomalies or intellectual disability: an evidence-based clinical guideline of the American College of Medical Genetics and Genomics (ACMG). Genet Med, 23 (2021)
  7. 3billion Blog. ACMG Recommends WES/WGS for Certain Patients