Approximate time required for this test:
3-6 Months (standard), 1 week (Rapid), 3 days (Ultra rapid)
Next generation sequencing (NGS) determines the order of nucleotides in a group of targeted genes (a gene panel), whole exome (all coding regions of the genome) or whole genome (all nuclear and mitochondrial DNA).
Compared to traditional Sanger sequencing, NGS technology allows the rapid sequencing of large amounts of DNA and is a more effective method of identifying the genetic basis of Mendelian disease. While NGS is a more efficient way of diagnosing genetic disease, there are some limitations. Sequence coverage, which refers to how much of the sequenced DNA aligns to (covers) the reference sequence, is incomplete in NGS and can cause false negative results. Coverage in whole exome sequencing (WES) is less than in whole genome sequencing (WGS) because only the coding regions are sequenced. Variations in NGS technique, such as short reads vs long reads, result in different coverage. NGS is also unable to identify certain types of genetic variants, such as triplet repeats and structural variants.
While sequencing DNA may be relatively straightforward, the interpretation of identified genetic variants isn’t always as easy, or even possible. Our ability to interpret NGS results is only as good as the current worldwide knowledge in variant databases.
DNA sequence data is analysed through a ‘pipeline’, a series of algorithms and statistical steps to check sequence quality, assemble data and compare to a reference.
The comparison of a DNA sequence to a reference sequence allows scientists to identify (call) variants. The reference sequence used for identifying variants is a sequence compiled from many human genome sequences and sourced from an international database.
The DNA sequence must pass Quality Control checks, including coverage, which needs to be sufficient to identify a variant relevant to the patient’s phenotype. Poor coverage limits interpretation and may require re-analysis or re-testing.
Sequencing may identify thousands of variants, so there needs to be a process of identifying only variants relevant to the patient phenotype. Applying gene lists and filters helps narrow the field of variants to find those of potential relevance. Filtering of sequence data allows analysts to ignore variants common in the population (single nucleotide polymorphisms or SNPs) and variants that are unlikely to cause genetic disease.
Variant identification & interpretation
Comparison of patient and reference DNA sequences reveals sites of variation; single nucleotide changes and small deletions, insertions or duplications. Variants in genes relevant to the patient phenotype are prioritised for analysis (curation) and classification, with evidence sourced from international databases, from benign to pathogenic.
Filtering allows you to focus on variants inherited in a recessive, dominant, autosomal or sex-linked pattern. Filtering can also exclude incidental finding of variants causing conditions unrelated to the patient’s phenotype.
An expert team including medical scientists, clinical geneticists and specialists then prioritise the variants based on rarity, potential to affect the protein and relevance to the patient’s phenotype. Prioritised variants are then curated.
Curation is a process of gathering evidence about a variant to determine whether it may be the cause of a patient’s condition/phenotype. Medical scientists and clinical geneticists consider multiple lines of evidence about the characteristics and location of a variant, using research and clinical data, population and disease databases and in silico (performed via computer) predictions tools. All evidence is weighed up to determine the potential for pathogenicity, i.e. is the variant a possible cause of the patient’s condition/phenotype.
The curated variants are classified on a 5 point scale – pathogenic, likely pathogenic, uncertain significance, likely benign and benign – described by the American College of Medical Genetics and Genomics (ACMG*). Evidence must be very strong and consistent to achieve a pathogenic classification.
*Reference: Richards et. al., Interpretation of sequence variants, Genetics in Medicine 2015; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4544753/
Case review / Multidisciplinary team
Variant classification is reviewed against the clinical information and patient phenotype. Variants that may explain the phenotype and clinical features are reported. Multidisciplinary team may include medical scientists, clinical geneticists, genetic counsellors and, ideally, the referring medical specialist.
A multidisciplinary case review supports clinicians to make informed decisions about the clinical relevance and clinical use of the genetic variants identified through testing. In the review meeting, variants with potential relevance to the patient’s condition, previously identified and curated by the medical scientist, are discussed in relation to the strength of the evidence, the clinical history and patient phenotype. The team may refer a variant for resequencing or further analysis or accept the variant classification and a report is prepared for the referring clinician. The final report may include recommendations for further analysis or segregation testing if appropriate.