TEST CODE: 008847

DIAGNOSTIC TEST FOR: WHOLE EXOME SEQUENCING

TURNAROUND TIME: 8-10 weeks

PREFERRED SPECIMEN: Blood (min. 1ml) in an EDTA tube or Extracted DNA, min. 2 μg in TE buffer or equivalent

ALTERNATE SPECIMENS: DNA or saliva/assisted saliva

Whole Exome Sequencing

Whole Exome sequence analysis of single patient cases, coupled with Whole Exome Deletion/Duplication analysis. The test allows detection of single-nucleotide and indel variants, as well as larger deletions/duplications.

Whole-exome sequencing (WES) is a robust and one of the most comprehensive genetic tests to identify the disease-causing changes in a large variety of genetic disorders. In WES, protein-coding regions of all genes (~20,000) of the human genome, i.e. exome, are sequenced using next-generation sequencing technologies. While the exome constitutes only ~1% of the whole genome, 85% of all disease-causing mutations are located there.

WES is most suitable for individuals with:

– a complex, unspecific genetic disorder with multiple differential diagnoses.

– a genetically heterogeneous disorder.

– a suspected genetic disorder where a specific genetic test is not available.

• unsuccessful previous genetic testing.

Test Strength and Limitations

The strengths of this test include:

– CAP and ISO-15189 accreditations covering all operations including all Whole Exome Sequencing, NGS panels and confirmatory testing

– CLIA-certified personnel performing clinical testing in a CLIA-certified laboratory

– Powerful sequencing technologies, advanced target enrichment methods and precision bioinformatics pipelines ensure superior analytical performance

– Our Nucleus online portal providing transparent and easy access to quality and performance data at the patient level

– Our publically available analytic validation demonstrating complete details of test performance

– ~1,500 non-coding disease causing variants in WES assay

– Our rigorous variant classification based on modified ACMG variant classification scheme

– Our systematic clinical interpretation workflow using proprietary software enabling accurate and traceable processing of NGS data

– Our comprehensive clinical statements

Test limitations

The technology may have limited sensitivity to detect variants in these genes.

This test does not detect the following:

– Complex inversions

– Gene conversions

– Balanced translocations

– Mitochondrial DNA variants (to include mtDNA analysis to your WES order, see Expansion Service)

– Repeat expansion disorders unless specifically mentioned

– Non-coding variants deeper than ±20 base pairs from exon-intron boundary unless otherwise indicated (please see above Panel Content / non-coding variants covered by the panel).

This test may not reliably detect the following:

– Low level mosaicism (variant with a minor allele fraction of 14.6% is detected with 90% probability)

– Stretches of mononucleotide repeats

– Indels larger than 50bp

– Single exon deletions or duplications

– Variants within pseudogene regions/duplicated segments

Test performance

We utilize whole exome capture technology and Next-Generation Sequencing methods to obtain clinical-grade WES data, maximizing coverage of clinically relevant genes.

– Highly uniform sequencing depth across all protein-coding genes of the genome

–Mean sequencing coverage on average 174x at guaranteed 100M sequencing reads

— On average, 99.4 % of base pairs in genes’ coding regions and selected intronic variants covered at least 20x

– Highly sensitive and specific detection of single-nucleotide variants and indels

— 99.7% sensitivity and >99.99% specificity for single-nucleotide variant detection within coding regions of genes and selected intronic variants.

— 97.0% sensitivity and >99.99% specificity for indel detection within coding regions of genes and selected intronic variants.

— Deletions up to 220bp detected, insertions up to 221bp

–  Assay performs with high precision

— Within-run precision (repeatability) 99.7%, intermediate precision (reproducibility) 99.7%

– Sensitive and specific detection of copy number variants (CNVs)

— Most of the single exon deletion events are detected and the sensitivity at five exon CNV level is >99% and specificity >99.9%. Segmentally duplicated genomic regions may have reduced sensitivity. The exact boundaries of the copy number aberration cannot be determined with this test.

Bioinformatics

Whole exome sequencing targets all protein coding exons and ± 20 base pairs from the exon-intron boundary. In addition, the test includes >1500 selected non-coding, deep intronic disease causing variants.

The sequencing data generated in our laboratory is analyzed with our proprietary data analysis and annotation pipeline, integrating state-of-the art algorithms and software solutions. The proprietary automated bioinformatics pipeline is streamlined to maximize sensitivity without sacrificing specificity. It enables detection of single-nucleotide and small indel variants from WES data in addition to large copy-number variants (≥1 exon level) when Plus analysis is requested. Quality control steps are included throughout to ensure the consistency, validity and accuracy of results.

– WES data are primarily analyzed for changes in genes that are known to be associated with human disease. We monitor recent literature and up-to-date databases to link variants in genes observed in patients with up-to-date information regarding the genes’ association with relevant diseases. To further aid the process of variant interpretation, observed variants are matched against a comprehensive set of databases of disease-related mutations, collected and curated in-house, and accessed from the public domain or licensed from commercial sources. We have incorporated a number of reference population databases and mutation databases such as, but not limited, to 1000 Genomes Project, gnomAD, ClinVar and HGMD into our clinical interpretation software to make the process effective and efficient. For missense variants, in silico tools such as SIFT, PolyPhen and MutationTaster are used to assist with variant classification. Splicing analysis is carried out by using Alamut Visual Software (SpliceSiteFinder-like, MaxEntScan, NNSPLICE, GeneSplicer).

Clinical interpretation

During the analysis of the WES data, we are looking for a genetic explanation for the patient’s symptoms. Therefore, analysis and reporting focus on variants that are directly related to the patient’s phenotype. This includes known/possibly disease-causing heterozygous variants in genes associated with autosomal dominant (AD) conditions, homozygous/compound heterozygous variants in genes associated with autosomal recessive (AR) conditions, or heterozygous/hemizygous/homozygous variants associated with X-linked disorders that are consistent with all, or a portion of, the patient´s phenotype as reported to the laboratory.

We use a variant-driven approach, often referred to as a ‘genotype-first’ strategy in the literature. This approach is considered to be one of the major benefits of WES and whole genome sequencing as it means that we do not pre-filter the sequencing data against predefined set of genes that are thought to be associated with patient’s disease but instead review all identified variants in all protein coding genes complemented by non-coding genes with a known association with human disease. The genotype-first approach considers that many patients referred for WES may have 1) an atypical presentation of a relatively well-known syndrome, 2) a genetically highly heterogeneous syndrome, 3) a very rare disease with a clinical picture that has not yet been well-established or 4) the possibility of multiple diagnoses that may confound the clinical presentation.

The clinical and family history of the patient, including symptoms, age of onset and prevalence and inheritance pattern of the disease are all taken into consideration. It is therefore important that the clinical and family history information provided is as detailed and complete as possible to ensure all relevant variants are reported. Carrier status of variants in genes not related to the patient’s phenotype are not specifically assessed and are not reported.

Analysis of the WES data first focuses on genes that have an established association with genetic disorders. The genes with a known clinical association include those curated by Blueprint Genetics (BpG) and included in BpG diagnostic panels. This list is supplemented with genes included in The Clinical Genomics Database and the Developmental Disorders Genotype-Phenotype Database (DD2GP). The total number of genes that are considered clinically associated is currently in the order of 3800 although this number is constantly changing.