Analysis methods

Technology

Blueprint Genetics uses a powerful molecular biology approach called next-generation sequencing (NGS) to analyze patient DNA. Changes to the DNA sequence (called variants) may be harmful and can result in severe medical conditions. Many hereditary diseases are caused by DNA variants originating in germ cells and subsequently present in all cells of the human body. Identifying disease-causing variants can lead to a diagnosis, determine the patient’s prognosis, and can help select the most appropriate treatments.

NGS allows for the comprehensive analysis of thousands of clinically relevant target genes in a timeframe that facilitates clinical action. NGS detects different types of variants such as nucleotide substitutions (point mutations) or the addition/deletion of nucleotides (insertions and deletions).

With targeted sequencing, it is possible to identify variants that have been previously linked to specific genetic disorders as well as novel variants in the disease-associated genes.

Targeted sequencing with NGS technology

In general, targeted sequencing refers to all technologies where only a selected portion of the genome is sequenced. A common, targeted sequencing approach is whole exome sequencing (WES) where specific molecular probes are used to capture DNA from the coding regions of >20,000 human genes. The process does not typically include the noncoding regions (>98.5%) of the human genome. In the context of specific phenotype-driven gene panels, a more targeted set of genes can be selected for sequencing. In targeted sequencing, the patient’s sample is prepared by adding sample-specific DNA adapters to the ends of the DNA fragments, capturing specific genomic regions by hybridization, and amplifying the sequencing libraries using PCR.

In the sequencing instrument, the sequencing library that has been prepared from the patient’s DNA is flushed through the sequencing flow cell. A flow cell is a fluidics device where the DNA fragments in the sequencing library are sequenced. Millions of DNA fragments attached to the flow cell surface form clusters of thousands of identical molecules. Each molecule on the flow cell is sequenced using sequencing-by-synthesis approach where fluorescently labelled nucleotides latch on to the ends of the growing DNA strands and images are captured. Sequencing proceeds in cycles where old labels are cleaved off, new labelled nucleotides are added and the imaging process is repeated. The images reveal all the bases and their order in each of the immobilized DNA fragments. The information content on the images is converted to raw data and extracted to text files.

Whole Exome Sequencing

Blueprint Genetics Whole Exome targets all protein-coding genes of the genome as well as a number of clinically relevant deep intronic variants. It has been developed to maximize diagnostic yields by generating high-quality and uniform sequencing data across the whole exome.

High-quality analysis

• On average, 99.4% of base pairs in the target region are covered at a sequencing depth of at least 20x
• Highly uniform sequencing depth across all target genes
• Single nucleotide variants (SNV): Sens 99.65% and Spec >99.99%
• Insertions, deletions and indels
• 1-10 bps: Sens 96.94% and Spec >99.99%
• 11-50 bps: Sens 99.07% and Spec >99.99%

Clinical Exome Sequencing

The Blueprint Genetics Clinical Exome Sequencing (CES) assay aims to cover all clinically relevant genomic targets in inherited diseases. It consists of >4000 genes, including all of our panel genes in addition to candidate genes not currently on the panels but curated by our clinical interpretation team based on data in the Clinical Genomic Database (CGD) and The Developmental Disorders Genotype-Phenotype Database (DDG2P). A number of candidate genes have also been included which are not present in CGD/DD2GP databases but where relevant publications are strongly suggestive of disease association and the gene has either high pLI (> 0.9) or high missense constraint (>3.0-4.0) in gnomAD have also been included. Our CES assay includes disease-causing, noncoding intronic variants associated with disease phenotypes, additional genomic targets for quality control of INDEL sensitivity, mitochondrial DNA, and pharmacogenetic targets.

The assay is based on high-quality, clinical-grade NGS that offers precise clinical diagnostic testing. All of our diagnostic panels and single genes are sectioned from the high-quality CES assay.

Blueprint Genetics utilizes high-quality custom capture technology and next-generation sequencing (NGS) methods to obtain deep and uniform data. All of our NGS technology is subjected to thorough quality control measures, after which raw sequence reads are transformed into variants by our proprietary bioinformatics pipeline.

High-quality analysis

• An average of 99.86% of base pairs in the target region are covered at a sequencing depth of at least 20x
• Highly uniform sequencing depth across all target genes
• Single nucleotide variants (SNV): Sens 99.89 % and Spec >99.99%
• Insertions, deletions and indels
• 1-10 bps: Sens 96.9% and Spec >99.99%
• 11-50 bps: Sens 99.13% and Spec >99.99%