Process

Ordering and sample preparation

The first step of the order process is to provide your patient’s medical history. You can do this through Nucleus, our online portal for ordering tests, tracking samples, and receiving test results. The entire process is transparent. You can see what is happening to your patient’s sample at each stage of the testing process through Nucleus.

When we receive the sample, we first check to ensure it meets the quality criteria required to produce an accurate result. If the sample is blood or saliva, it’s run through an extraction robot to acquire the DNA. We then perform a series of molecular biology techniques to prepare the sample DNA for sequencing.

First, the DNA is sheared into fragments, each comprised of about 1,000 nucleotides. Next, a synthetic DNA sequence is added to both ends of each DNA fragment during adapter ligation. These synthetic ends allow the DNA fragment to interact with the sequencing system, while also tagging the DNA to a specific patient sample. Finally, the adapted DNA fragments are copied multiple times in a process called enrichment, which produces enough of the necessary DNA for successful sequencing.

All of the methods used in this preparatory phase are tested and trusted methods of DNA sequencing preparation. A reference DNA is also prepared alongside the samples to serve as the control during sequencing. Since the sequencing results for this reference DNA are known, it ensures high-quality, reliable results.

 

Targeted sequencing with NGS technology

In general, targeted sequencing refers to all technologies where a selected portion of the genome is sequenced bringing cost effectiveness into the process. The most common targeted sequencing approach is whole exome sequencing (WES) where specific molecular probes are used to capture DNA from coding regions of >20,000 human genes. The process leaves out non-coding regions (>98.5%) of the human genome. In context of specific phenotype driven gene panels, the target genes may be selected before or concomitantly with sequencing or sliced out from WES data. In WES, 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, sequencing library that has been prepared from the patient’s DNA is flushed through the sequencing flow cell. 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 products target all protein-coding genes of the genome. They have been developed to maximize diagnostic yields, first of all, by generating high-quality and uniform sequencing data across the whole exome. Blueprint Genetics utilizes high-quality exome capture technology and next-generation sequencing methods to obtain deep and uniform, clinical-grade whole-exome sequencing data. Each exome is subjected to thorough quality control measures, after which raw sequence reads are transformed into variants by our proprietary bioinformatics pipeline. Finally, the variants are analyzed and interpreted in-house by our team of geneticists and clinicians.

Data analysis and interpretation

Sequencing creates a massive amount of raw data, which requires heavy processing to become clinically relevant information. The proprietary automated bioinformatics pipeline developed and used by Blueprint Genetics produces fast, reliable results. All of the software, equipment, and algorithms used are industry-standard in the field of molecular genetics, but specific parameters have been adjusted and other customizations have been made to improve data processing and analysis.

The first step in analyzing the results is quality filtering on the raw sequencing reads. This allows any uncertain nucleotides to be trimmed out of the data pool. Once the highest-quality reads have been isolated, they are aligned with the human reference genome so variant calling can begin. Variants are differences from the reference genome – hundreds to thousands may be found in any patient’s DNA depending on the target size, but most of the variants are clinically irrelevant to the suspected diagnosis.

To aid in variant interpretation, Blueprint Genetics uses a comprehensive, proprietary mutation database containing curated disease-related variants. This database is comprised of information from more than 2,500 scientific publications and publicly available genetic databases. The output of the bioinformatics pipeline is a Sequence Analysis Report, which contains the detailed results of the test, related literature links, the pathogenicity evaluation, and the quality assessment.

A comprehensive clinical report

The final phase of Blueprint Genetics’ all-inclusive process is the geneticist’s statement, which is supported by the insight of a specialized clinician. For positive results, the report generally includes valuable insights into potential treatments while highlighting any risks the disease presents to the patient. The concluding statement is delivered to you through Nucleus, by mail, or by fax, depending on your preferences.

If the results are negative, the statement helps move toward a potential future diagnosis by ruling out one or more possibilities. The geneticist’s statement may also include recommendations for a different, more comprehensive panel when deemed beneficial. In this event, Blueprint Genetics generally does not require a new sample to run the different panel, so it’s easy for you to order a new panel if needed.