METHODOLOGY HIGHLIGHTS

• Clinical Rule-out Sequencing
• Sequence and Copy Number Analysis for Every Gene and Every Exon on Every Panel
• Sensitivity Outperforms “Gold Standard” Sanger and MLPA
• All Findings Confirmed using Classical Techniques
• Breakthrough in Cost-Effectiveness

TECHNICAL INFORMATION

The following information outlines the techniques and benefits of tests at LHSC.

Techniques:

All coding exons and 20 bp of flanking intronic sequence are enriched using an LHSC custom targeted hybridization protocol (Roche Nimblegen), followed by high throughput sequencing (Illumina). This chemistry and analysis pipeline provides a highly sensitive and specific detection of sequence and copy number alterations in a single assay that exceeds the previous gold standard of Sanger sequence and MLPA. Sequence variants and copy number changes are assessed and interpreted using clinically validated algorithms and commercial software (SoftGenetics: Nextgene, Geneticist Assistant, Mutation Surveyor; and Alamut Visual). All genes have >1000x mean read depth coverage, with a minimum 200x coverage at a single nucleotide resolution. Mitochondrial DNA testing is validated for heteroplasmy detection sensitivity of 2-5%. All variants interpreted as either ACMG category 1, 2, or 3 (pathogenic, likely pathogenic, VUS; PMID: 25741868) are confirmed using Sanger sequencing, MLPA, or other assays. ACMG category 4 and 5 variants (likely benign, benign) are not reported, but are available upon request.

Analysis includes copy number assessment for the mitochondrial DNA deletion syndrome (Kearns-Sayre syndrome). The focus of the LHSC custom panels is to provide a “rule out” assay for every gene included in a given panel, and not simply a screen of genes.

Benefits of testing:

LHSC Molecular genetics panels are developed in close consultation with clinician groups to ensure the panels provide clinically actionable results which will directly impact medical management recommendations and disease risk assessment.

It will allow the assessment of an individual’s genetic test results which, in combination with their personal and family cancer history, will assist the clinician in determining an optimum pathway for their patient’s immediate medical and/or surgical management along with clinical follow up. Individuals identified as carrying highly penetrant, deleterious gene mutations, will be counselled on the potential effects of these mutations, and could be offered appropriate specialist medical referral for optimum ongoing management.

Turnaround time:

4 – 6 weeks

Mutation detection rate:

The LHSC NGS protocol is predicted to detect in excess of 99% of mutations present within the genes listed. Mutation detection rates in the patient are dependent on the clinical presentation including age of onset, specific diagnostic features, and family history. Detection rates are specific to each panel but typically fall into the 10-20% range in clinically affected patients.

Specimen requirements:

• 4 ml EDTA blood
• 1 – 2 ug DNA

References:

Clinical NGS Pipeline outperforms a combined approach using Sanger sequencing and MLPA in targeted gene panel analysis (Schenkel et al, J Mol Diagn. 2016 Sep; 18(5):657-67)