Exome and Genome Sequencing for the Diagnosis of Rare Diseases

Background: Genetic changes in the germline are the main cause of rare diseases (RD) and represent a significant disease burden in the population. Rapid and comprehensive genetic diagnosis is the key to clinical management.

Methods: Whole-genome sequencing was used to diagnose RD in a clinically heterogeneous patient cohort representative of real-world clinical practice. Three subgroups were distinguished based on genetic pre-diagnosis. 963 genomes were sequenced (360 index individuals from different families and 603 family members). The families received genetic counseling and were asked about their acceptance of the diagnostic test.

Results: The diagnosis rate in the overall cohort was 30% (95% confidence interval: [25.3; 34.7%]; 108/360). In 2.2% ([0.7; 3.7%]; 8/360) of cases, variants were found that would not have been detectable in the exome, corresponding to 7.4% ([2.4; 12.4%]; 8/108) among the detected cases in a direct comparison of “exome versus genome.” The acceptance of genome sequencing was very high when embedded in human genetic counseling.

Conclusion: Genome sequencing is an effective way to diagnose RD. Genome data provide a comprehensive basis for continuous re-evaluation and are superior to exome data in this respect. The integration of genome sequencing into standard care is now under evaluation as part of the nationwide “Model project genome sequencing” in Germany.

Cite this as: Elbracht M, Krause J, Mattern L, Güzel N, Lischka A, Suh DSJ, Knopp C, Bourgeois MG, Beijer D, D’Augello S, Haag N, Rüdebusch J, Perchalla E, Lausberg E, Eggermann K, Meyer R, Kraft F, Begemann M, Eggermann T, Kurth I, on behalf of the members of the study group: Exome and genome sequencing for the diagnosis of rare diseases. Dtsch Arztebl Int 2026; 123: 184–8. DOI: 10.3238/arztebl.m2026.0030

The significance of genetic variations in the development of a wide variety of diseases is becoming increasingly clear. This makes the need for comprehensive genetic testing all the more apparent. Molecular findings can provide the basis for targeted treatment decisions and preventive measures and quite often put an end to a stressful, and often lengthy, investigation into the underlying cause (1, 2). The identification of genomic aberrations is particularly crucial for the diagnosis and therapeutic management of rare diseases (RDs) and oncological diseases (ODs) (3, 4).

By definition, RDs affect fewer than 1 in 2000 individuals; there are more than 8000 distinct entities altogether, so the cumulative prevalence is estimated at around 3.5 to 5% of the general population. This corresponds to a figure of approximately four million affected individuals in Germany. In around 80% of cases, RDs have one single genetically determined cause and are classified as monogenic. The average time to diagnosis of an RD is five years and is often preceded by multiple misdiagnoses and costly and stressful additional diagnostic investigations (5). This, together with the large number of potentially eligible genes, supports early and comprehensive molecular genetic testing to improve patient care (6, 7).

In recent years, various methods of high-throughput sequencing (next-generation sequencing, [NGS]) have gained widespread recognition in genetic testing (Box 1). Exome sequencing is one of these methods and allows the parallel analysis of all protein-coding gene segments and their adjacent regions, which together comprise only around 2% of the human genome, however (8). Genome sequencing extends beyond this assay, since it captures the entire genetic makeup – including regulatory, non-protein-coding, and structurally altered regions, most of which are not typically included in the exome. With this in mind, the question arises regarding the current and future roles of genome sequencing in human genetic services.

Box 1

Overview of the different sequencing modalities and techniques

Project description and aims

As part of a single-center study conducted in 2024 at the Uniklinik RWTH Aachen, the benefit of genome sequencing for establishing RD diagnoses was examined. Sequencing was embedded in a structured genetic counseling service, both before and after the laboratory analysis, and was supplemented by an anonymized patient questionnaire. The aim of the study was to evaluate the diagnostic benefits of genome sequencing, to systematically record patient acceptance, and to analyze its feasibility in clinical practice across a wide range of genetically determined conditions.

Methods

In this single-center pilot study, 963 individuals were assessed using genome sequencing (360 index patients with a suspected RD, 603 family members). The overall cohort was divided into three subgroups: “no prior genetic testing” (n = 240 index patients), “prior genetic testing without NGS” (n = 51 index patients), “prior exome analysis” (n = 69 index patients). In addition, a clinical classification into four indication groups was undertaken (syndromic disorders [syndromic], retinal diseases [retina], neurological disorders [neurological], and hereditary cancer predisposition syndromes [cancer predisposition], Box 2). The analyses were performed predominantly as trio genome sequencing. Before and after genetic counseling and after communication of the results, index patients were questioned anonymously with respect to their stress, expectations, and benefits. Parents and caregivers were interviewed when children and persons lacking legal capacity were involved. Questionnaires were completed before and another after genome sequencing for each index patient. Sequencing was performed as short-read sequencing (Illumina NovaSeq 6000) and analyzed using DRAGEN, Emedgene, and the institute’s own pipeline (9). Phenotypes were coded using the Human Phenotype Ontology (HPO) and used for phenotype-assisted, AI-based variant prioritization (10). The project had received ethical and data-protection approval (EK21–445).

Box 2

Genome study design*

eBox

Members of the study group (collaborators)

Results

The findings of the study were assessed with respect to diagnosis rates, benefits of genome sequencing, particularly in comparison with exome sequencing, and to patient acceptance of the comprehensive genetic data acquisition.

General diagnosis rates

Classification of the variants was undertaken in accordance with the ACMG criteria and the “ACGS Best Practice Guidelines for Variant Classification in Rare Disease 2020”, with the caveat that, as yet, there are no established comprehensive criteria available for genome evaluation (11). Four diagnostic categories were defined: 1. “secure diagnosis” (variant classified as pathogenic or likely pathogenic); 2. “probable diagnosis” (variant formally classified by ACMG criteria as a variant of uncertain significance, VUS) but deemed highly plausible from a clinical genetics perspective based on phenotype, segregation, and additional criteria, such as variant type, so-called “hot VUS”); 3. “possible diagnosis” (variant with potential clinical relevance, requiring additional analyses for confirmation, so-called “warm VUS”), and 4. “unsolved cases” (no plausible variant, and including tepid, cool, cold, ice-cold VUS).

The diagnostic yield (cases in diagnostic categories 1 and 2) of genome sequencing was 30% (95% confidence interval: [25,3; 34,7]; 108/360). When analyzed according to prior diagnostic testing, the following diagnostic yields were observed: “No prior genetic testing”: 30.4% ([24.6; 36.2]; 73/240), “Prior diagnostic testing without NGS”: 33.3% ([20.3; 46.4]; 17/51), “Prior exome analysis“: 26.0% ([15.7; 36.5]; 18/69) (Table 1). In addition, a “possible diagnosis” (diagnosis category 3) was established in 10% of the index patients [6,9; 13,1] (eTable 1).

Table 1

Diagnostic yields: comparison of exome and genome analysis, stratified by prior genetic testing

Benefits of genome sequencing

A key aim of the study was to analyze the additional diagnostic benefits of genome sequencing in comparison with established exome sequencing. An assessment was therefore conducted to determine whether a diagnosis established by genome sequencing could also have been reached by an analysis of the existing genomic data limited to the exome (exome extract). (Table 1). Based on all solved cases, this would have been possible in 92.6% [87.7; 975]. Conversely, in 7.4% [2.4; 12.4] of the solved cases, the molecular diagnosis was only reached by genome analysis.

Variant types

A breakdown of variants classified as pathogenic or likely pathogenic by type revealed that 62% were small exonic variants (single nucleotide variants, SNV) (Table 2, Box 1).

Table 2

Breakdown of causal genetic variants of the cases by types*

The next most common variant types were copy number variants (CNVs) and structural rearrangements, such as insertions and deletions (indels), which are detected systematically more easily and more reliably by genome sequencing than by exome sequencing. CNVs were detected in 12.4% of cases, indels in 10.9%.

In recent years, numerous new disease entities have been reported that are attributable to repeat expansions which are difficult or impossible to detect by exome sequencing (12). Such repeat expansions were identified as causative variants in 7% of the solved cases (Table 2).

A splice variant (+/− 15 base pairs from the exon boundaries) was found in approximately 5% of the patients. In one case, a so-called deep intronic splice variant beyond this range was identified, which, however, was only to be diagnosed by genome sequencing and not captured by exome sequencing. In addition, further extragenic pathogenic variants, for example, in non-protein-coding RNAs (such as RNU genes), were also detectable by genome sequencing.

Patient questionnaire on genome sequencing

A questionnaire was given to each index family before and again after genome sequencing. Evaluation of the patient questionnaire on genome sequencing (eTable 2) shows that the lack of a confirmed genetic diagnosis poses a significant burden for many participants (72%), based on participation rates of 77.2% before (n = 278) and 34.7% after (n = 125) among index patients. After receiving appropriate instructions and explanations, the vast majority (96%) rated the methods used as easy to understand and, in hindsight, felt the testing procedure was less complex than expected. Thirty-seven percent of respondents said they had feared the possible results. At the same time, most of them expected real benefits, especially more targeted information about trials and ongoing research projects (89%), improved prognostic assessment and treatment planning (98%), and, to a lesser extent, family planning (41%). This last percentage should be interpreted in the context of the large proportion of participants under the age of 18 (48%).

eTable 2

Results of the anonymized genome sequencing questionnaire

Regarding the questionnaire after the diagnostic testing, approximately 60% of the respondents said they intended to use the results to seek targeted information on further trials and research projects, to adapt future medical decisions, or to take the results into account when making family planning decisions.

Ninety-four percent of the respondents were satisfied with how the genome study had been conducted (80%: strongly agree; 14%: agree). However, 20% (7.2%: strongly agree; 12.8%: agree) reported being unsettled by the results and feeling more distressed than before the test (eTable 2). Responses were provided anonymously, so it is not possible to draw any conclusions regarding correlations between responses and the recorded genetic findings. It is therefore not possible to assess self-selection bias. The questionnaires for children and persons lacking legal capacity were completed on their behalf by parents or caregivers, and, together with the approximately 35% response rate following genome sequencing, this limits the interpretability of the results.

Discussion

Genome sequencing analyzes the entire genome and is therefore the most comprehensive method of genetic testing currently available. It has the potential to achieve the highest diagnostic yield in patients with rare diseases. At the moment, the obstacles preventing its nationwide implementation are mainly related to financing this health care provision, the acquisition and maintenance of high-performance sequencers, the need for computing and storage resources, and the requirements for bioinformatic analysis and human genetic interpretation. The latter also includes an increased need for qualified genetic counseling before and after diagnostic testing and requires more interdisciplinary management within specialized departments and medical practices.

In clinical practice, patients present with a wide range of RDs, making genome sequencing and genetic assessment suitable for a heterogeneous spectrum of indications (13). The inclusion criteria of the present study were therefore defined appropriately broadly. The diagnostic yield (solved + likely solved cases) was 30%, plus a further 10% with a possible diagnosis.

A recently published study involving 822 families established a molecular diagnosis in 29.3% of cases with the use of genome sequencing. Of these families, 8.2% had variants identified only by genome sequencing – findings that closely match the results of the present study (14). A meta-analysis of pediatric and adult patients with RDs across diverse populations showed no significant differences in the pooled diagnostic yield between genome and exome sequencing, but high-quality studies demonstrated a significantly higher clinical benefit of genome sequencing (15).

Exome-based studies involving selected cohorts – for example, pediatric populations, intensive care units, or with selected indications – achieved high diagnostic yields of over 30% to, at times, over 50% (including, for example, one of our own studies) (16), but these results are often based on prior clinical selection with an anticipated high proportion of monogenic diseases (17, 18). Among patients with cancer predisposition syndromes, our study identified a proportion of 14.3% of cases that were likely attributable to a monogenic cause, in line with the current literature. This share is lower than that of the other study groups examined here, yet substantial, because a diagnosis is crucial for the direct therapeutic management of the index patient, in particular for cancer diseases, and at the same time for the counseling and intensified surveillance of other family members at risk.

The results of the present study demonstrate that, in a direct comparison of exome and genome sequencing, causative genetic findings can be detected by genome sequencing alone in approximately 7% of cases. Conservative comparison criteria were deliberately used for the direct comparison – such as the assumption that small CNVs would also be reliably detected by exome sequencing, which is often not the case in actual practice, however. Therefore, the actual diagnostic gap between exome and genome sequencing is most likely already wider than the 7% stated here. The present study addresses the use of short-read sequencing and evaluation based on current knowledge of genome-wide pathogenic variants. In the long term, long-read genome sequencing could, among other things, increase the detection rate of pathogenic structural variants and repetitive regions even more reliably (19).

A major benefit of genome sequencing over exome sequencing lies in the wealth of data it generates: the totality of the identified genetic information allows continuous, comprehensive reanalysis of previously unsolved cases. New disease-relevant gene-phenotype associations and advances in bioinformatics and databases (for example, ClinVar) will, in the future, allow additional diagnoses without the need for repeat sequencing. However, the current healthcare system in Germany lacks appropriate billing codes for reanalyses. A study recently published by the Solve-RD Consortium identified 8.4% of the previously undiagnosed rare diseases through systematic reanalysis of exome data – this rate increased to 12.6% when specialized expert panels were involved (20). At least comparable development may be expected in genome sequencing, especially if the data are systematically enriched with clinical information. International projects, such as Genomics England, already show how the structured combining of clinical and whole-genome data can improve healthcare delivery (21, 22). The studies refer to subsequent changes in medication, additional surveillance, inclusion in clinical trials, as well as reproduction and family planning.

In Section 64e of the German Social Code Book V (SGB V), the German Health Care Development Act defined a pilot project for comprehensive diagnostics and treatment selection using genome sequencing for both rare and oncological diseases. Since September 2024, genome sequencing has been conducted and evaluated at qualified university hospitals as part of a structured clinical management pathway over a period of five years. A central element here includes interdisciplinary case conferences involving centers for rare diseases and centers for personalized medicine. The integration of clinical and genomic data is achieved within a central data infrastructure to facilitate, amongst other things, medical care and to provide a platform for reanalysis. During the first two assessment periods, genomic diagnostics, including all the associated processes described above, are funded for a limited number of patients at 8000 euros per case. The pilot program presents an opportunity to establish genome sequencing beyond pilot studies as a standard option in routine healthcare, consistent with modern genomic medicine.

Limitations

The present study reports initial findings on the diagnostic and clinical benefit of genome sequencing in standard healthcare in Germany. However, it is a single-center study with a limited number of cases. In addition, a broad spectrum of rare diseases was deliberately included, as is typical of everyday clinical practice. However, this heterogeneity, with its variety of pre-diagnostic genetic tests, limits the interpretability of the diagnostic yield for specific indication groups. The present study did not address the direct impact of the diagnosis on individual clinical management.

Conclusions

The present study demonstrates the diagnostic yield of exome and genome sequencing for rare diseases. As knowledge of the significance of non-protein-coding genetic variants increases and opportunities for reanalysis of comprehensive genomic data grow, the clinical value of genome sequencing relative to other modalities is also likely to increase further. The findings of this pilot study emphasize the medical relevance of long-term, interdisciplinary integration of genome sequencing into standard healthcare.

Acknowledgments
We would like to thank Sebastian Giesselmann, Elvira Golz, Stephan Klinkenberg, and Ronja Wahl for their excellent technical support in conducting the genome sequencing. The study was in part supported by consumables and access to Emedgene analysis software provided by the Illumina company. We also express our thanks to Dr. Sven Schaffer for his critical review of the manuscript, Laura Bell from the AVMZ Audiovisual Media Center of the Uniklinik RWTH Aachen for her assistance in presenting the study design, and especially to all study participants.

Conflict of interest statement
The authors declare that no conflict of interest exists.

Manuscript received on 18 August 2025, revised version accepted on 19 February 2026.

Translated from the original German by Dr. Grahame Larkin.

Corresponding authors:
Prof. Dr. med. Miriam Elbracht

Prof. Dr. med. Ingo Kurth

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