NGS (Next Generation Sequencing)

NGS genetic testing is a modern next-generation sequencing method that enables a detailed examination of genetic material. This technology uses advanced bioinformatics tools to analyze sequenced DNA. It converts the data into reliable results that geneticists can use for diagnosis and further research.

What is NGS?

NGS (Next-Generation Sequencing) is an advanced gene sequencing technology that allows for rapid and comprehensive examination of genetic variations associated with diseases or other biological phenomena. This method enables the simultaneous sequencing of millions of nucleic acid fragments. This represents a significant advancement over earlier techniques, such as Sanger sequencing. NGS became commercially available in 2005 and has since significantly enhanced the capabilities of genetic diagnostics.

NGS provides insights into various genomic features. This includes single nucleotide variants (SNVs), structural variants, gene copy number variations, and RNA fusions. Additionally, NGS offers greater accuracy, requires smaller sample sizes, and can detect variants present at low allele frequencies.

The speed, capacity, and accuracy of NGS technology have revolutionized genetic testing. It has enabled new applications in genomics, clinical research, reproductive health, and forensic, agricultural, and environmental sciences. Sanger sequencing, still valuable for analyzing a smaller number of genes, is often used to confirm results obtained from NGS methods.

How is Next Generation Sequencing performed?

Next Generation Sequencing involves several steps that are consistent across different sequencing technologies. The process begins with sample preparation, followed by clonal amplification, sequencing, and concluding with data analysis.

• NGS library preparation: The first step in the NGS process is to prepare a “library” of sequences from a DNA or RNA sample. After isolating nucleic acids from the sample, they are fragmented into smaller pieces (100–800 base pairs) for sequencing. These fragments are tagged with specific adapters that allow them to bind to the surface of sequencing plates and be amplified. In some cases, fragments are also labeled with unique molecular “barcodes.” This allows multiple samples to be sequenced simultaneously in one run, saving time and resources.
• Clonal amplification: After library preparation, the DNA fragments need to be attached to a solid surface and clonally amplified to increase the signal that will be detected during sequencing. Each unique DNA molecule in the library binds to the surface of a bead or plate, where it is then amplified via PCR to create a cluster of identical copies. This process ensures a strong enough signal for detection during sequencing.
• Sequencing: The prepared fragments are placed on a sequencing platform, where a large number of fragments are sequenced simultaneously. During this process, nucleotides are added to the fragments one by one, and each added nucleotide is detected based on the emitted light or another specific signal for that nucleotide.
• Data analysis: After sequencing, the generated data is processed using bioinformatics tools. These tools assemble the sequences of the fragments and map them to a reference genome. They analyze variations such as single nucleotide variants, deletions, duplications, and other genetic changes. Finally, the results are interpreted, and reports are generated.

Because of the complexity of the data and the algorithms involved, analyzing NGS results typically requires specialized bioinformatics tools and experts. However, tools have been developed to make analysis more accessible for those without advanced bioinformatics training.

What is NGS used for?

NGS is used in a wide range of fields, including genetic and genomic testing. It is also applied in research across various scientific disciplines.

Prenatal diagnosis

NGS enables non-invasive genetic testing during pregnancy by analyzing cell-free fetal DNA from the mother’s blood. This testing can detect genetic disorders in the fetus. Prenatal genetic analysis is essential for early diagnosis and provides expectant parents with the information needed to make informed decisions.

Next Generation Sequencing in Belgrade is available at IntroLab, offering reliable results and the capability for early detection of potential genetic problems.

• BASIC test: T 21, 18, 13 + gender
• PLUS test: T 21, 18, 13 + sex chromosomes + gender
• EXPERT test: T 21, 18, 13 + sex chromosomes + DiGeorge Syndrome + gender
• PREMIUM test: T 21, 18, 13 + sex chromosomes + microdeletions + gender
• ALL CHROMOSOMES test: All chromosomes + sex chromosomes + deletions and microdeletions + gender
• SPECIAL test: T 21, 18, 13 + sex chromosomes + microdeletions + 100 hereditary diseases + gender
• SPECIAL + SMA test: T 21, 18, 13 + sex chromosomes + microdeletions + 100 monogenic diseases + SMA + gender

Oncology

In oncology, NGS technology enables the precise identification of specific mutations in tumors, which aids in selecting the most effective therapy for each patient. Genetic testing of tumors can improve treatment outcomes by allowing therapy to be tailored based on the tumor’s unique genetic profile.

Diagnosis of rare diseases

One of the most significant applications of NGS technology is in genetic testing for rare diseases. These diseases are often caused by genetic mutations that are difficult to detect using traditional methods. NGS diagnostics provide a detailed examination of the genome, which can lead to the identification of rare genetic variants responsible for these diseases.

Forensic science, microbiology, and ecology

Beyond medical applications, NGS is also utilized in forensic science to identify individuals based on biological traces, in microbiology for analyzing the microbiome, and in ecology to study biodiversity.

Who can benefit from NGS genetic testing?

NGS genetic testing can be valuable for a wide range of individuals and groups:

• Pregnant women and couples planning a pregnancy: Non-invasive prenatal testing using NGS can detect genetic abnormalities in the fetus, such as Down syndrome and other chromosomal disorders.
• Patients suspected of having genetic disorders: NGS aids in the precise identification of rare or inherited genetic disorders, allowing for more accurate diagnosis and personalized therapies.
• Cancer patients: NGS identifies specific mutations in tumors, enabling personalized treatment based on the tumor’s genetic profile.
• Individuals with a family history of genetic diseases: Preventive NGS testing can uncover hereditary risks, allowing for informed health decisions.
• Couples dealing with infertility or recurrent miscarriages: NGS helps identify genetic causes of infertility, leading to improved treatment options.
• Healthcare professionals and researchers: NGS results support diagnosis, therapy planning, and research into the genetic basis of diseases.

NGS diagnostics at IntroLab

At IntroLab, we provide genetic tests using advanced NGS technology, in collaboration with renowned international laboratories. Our panels are carefully designed to meet the specific needs of our clients, with the option for additional analysis based on a doctor’s recommendation.

Some of the available panels include:

• Autoinflammatory diseases panel: Analyzes 52 genes associated with autoinflammatory conditions, enabling accurate diagnosis and management of these disorders.
• Cardiomyopathy panel: Covers 173 genes related to cardiomyopathies, helping to identify causes and plan treatment for heart diseases.
• Arrhythmia panel: Analyzes 58 genes associated with various types of arrhythmias, allowing for precise diagnosis and treatment planning.
• Marfan syndrome panel: Covers 34 genes related to Marfan syndrome, providing a detailed evaluation necessary for diagnosis and monitoring of the disease.
• Epilepsy panel: Analyzes 480 genes linked to epilepsy, allowing for the identification of genetic causes and the customization of therapies.
• Clinical exome sequencing: Targets 85% of known genetic variants in exonic regions, useful for the rapid diagnosis of rare diseases and exploring the genetic causes of various disorders.
• Molecular karyotype (CGH array): Detects large and small chromosomal abnormalities, including microdeletions and microduplications, which can be associated with serious clinical conditions such as DiGeorge, Angelman, and Prader-Willi syndromes.

Sample for analysis

For NGS genetic analysis, an EDTA blood sample is used. This type of sample is stable and allows for precise analysis of genetic material. If necessary, depending on the selected analysis, other biological samples (such as saliva, buccal swabs, or tissue) can also be used.

Turnaround time for results

At IntroLab, the turnaround time for NGS analysis results ranges from 4 to 6 weeks, depending on the complexity of the test and the specific analyses being performed.

Are NGS results reliable, and how are they interpreted?

NGS test results are highly reliable due to the precision of this technology. At IntroLab, each analysis comes with a personalized report from a geneticist who provides a detailed interpretation of the results. Additionally, patients can schedule a genetic counseling session at our lab, where they will receive further support and guidance regarding their results and possible next steps.