NGS - OrbitoAsia Diagnostics

Genomics

Next Generation Sequencing (NGS)

Introduction

Next-Generation Sequencing (NGS) represents a transformative advancement in genomics, allowing for rapid, cost-effective, and high-throughput sequencing of DNA and RNA. Unlike traditional Sanger sequencing, NGS facilitates massively parallel sequencing, enabling the analysis of millions of sequences simultaneously and providing comprehensive genomic data.

NGS Platforms and Technologies

1. Genexus System

Features

Automation: Minimizes manual intervention, reducing errors and hands-on time.
Speed: Provides results in as little as one day.
Flexibility: Applicable to a range of fields including oncology and infectious diseases.
Usability: Designed for ease of use with a straightforward interface, suitable for various laboratory sizes.

2. Thermo Fisher Ion S5 Plus Sequencer

Features

Scalability: Adapts to small and medium-sized sequencing projects.
Speed: Quick sequencing runs, ideal for urgent applications.
Accuracy: High sensitivity for variant detection.
Broad Application Range: Supports diverse applications including targeted gene panels and exome sequencing.

3. Nanopore Sequencing (Oxford Nanopore Technologies)

Features

Long Reads: Provides high-resolution sequencing of long DNA/RNA fragments.
Real-Time Data: Allows for immediate data analysis.
Portability: Devices like MinION are portable for flexible usage.
Applications: Useful for metagenomics, transcriptomics, and pathogen detection.

4. Illumina Sequencing Platforms

Features

High Throughput: Platforms like NovaSeq offer extremely high throughput for large-scale projects.
Accuracy: Renowned for precise sequencing data.
Versatility: Platforms like MiSeq and NextSeq cater to a wide range of sequencing needs.
Applications: Extensively used in research areas such as oncology and agriculture.

Benefits of NGS

High Throughput: Sequences millions of fragments simultaneously.
Cost-Effective: Lower cost per base compared to traditional methods.
Speed: Faster processing times.
Accuracy: High sensitivity for detecting genetic variations.

Non-Invasive Prenatal Screening (NIPS)

Non-Invasive Prenatal Screening (NIPS) is an advanced method that analyzes cell-free fetal DNA in a pregnant woman’s blood, offering a safer and highly accurate alternative to invasive procedures like amniocentesis and chorionic villus sampling (CVS). With an accuracy rate exceeding 99.9%, NIPS poses no risk to the fetus compared to invasive tests.

Eligibility and Restrictions

Suitable for: Pregnancies from 10 weeks gestation, including singleton, twin, IVF, and surrogate pregnancies.
Not suitable for: Women with cancer, those who have had organ transplants, possess chromosomal imbalances, received heterologous cell transfusions recently, or have complete or partial monosomy X (Turner’s Syndrome).
Note: As a screening test, NIPS requires confirmation of high-risk results through invasive procedures like amniocentesis.

Who needs to get tested?

NIPS is recommended for all pregnant women, regardless of age, seeking information about their baby’s development. It is particularly advised for:

Women over 30 years old.
Cases with abnormal serum screening results.
Identified ultrasound abnormalities.
Family history of chromosomal conditions or birth defects.
Previous children with chromosomal disorders or a history of infertility or pregnancy loss.

Choosing Between Basic and Advanced NIPS

Consider personal risk factors, budget constraints, and clinical advice when deciding between basic and advanced NIPS. Basic NIPS screens for common chromosomal abnormalities, while advanced NIPS includes additional conditions such as microdeletions and rare trisomies.

Exome Sequencing

Exome sequencing is a genomic method that targets the protein-coding regions of the genome, known as exons. Although exons make up just 1-2% of the human genome, they account for approximately 85% of known genetic variants related to diseases. This technique provides a detailed examination of the exome, making it a powerful tool for detecting genetic mutations associated with various disorders.

Importance of Exome Sequencing

Disease Relevance: Since most genetic disorders are caused by mutations in exons, exome sequencing is crucial for clinical diagnostics.
Cost-Effectiveness: By focusing on the most relevant parts of the genome, exome sequencing is more affordable than whole-genome sequencing while still delivering extensive genetic information.
Research and Diagnosis: It enhances the discovery of new disease-related genes and helps diagnose complex genetic conditions.

Whole Exome Sequencing (WES)

WES examines all coding regions and splice junctions of the genome, providing a thorough assessment of protein-coding genes. It is particularly effective for identifying disease-related mutations.

When to Consider WES

For individuals with unexplained genetic disorders or inconclusive prior tests.
Those with a family history of cancer or genetic conditions.
Couples planning to have children with known genetic risks.
Cases involving fetal DNA for prenatal diagnosis.
Individuals with unexplained developmental or neurological conditions.

Clinical Exome Sequencing (CES)

CES focuses on a specific subset of clinically relevant genes, offering a more targeted approach compared to WES. It is useful for diagnosing disorders with known genetic links.

When to Consider CES

Individuals with suspected genetic conditions or previous inconclusive tests.
Patients with a family history of cancer or genetic disorders.
Couples planning to have children with known genetic risks.

BRCA1 and BRCA2

BRCA1 and BRCA2 testing involves analyzing specific genes known for their role in repairing DNA and maintaining genomic stability. These tumor suppressor genes are crucial for preventing cancer, and mutations in them significantly increase the risk of developing breast, ovarian, and other cancers.

Increased Cancer Risks

Women: Those with BRCA1 or BRCA2 mutations are at a higher risk of breast and ovarian cancers. Additionally, these mutations can increase the risk of cervical, uterine, colon, pancreatic, gallbladder, bile duct, stomach cancers, and melanoma.
Men: BRCA1 and BRCA2 mutations elevate the risk of breast, pancreatic, testicular, and prostate cancers.

Although BRCA1 and BRCA2 mutations are linked to only about 5% of breast cancers and 10-15% of ovarian cancers, they are significant risk factors.

When to Consider Testing

A family history of breast cancer, particularly with associated cancers such as prostate, ovarian, melanoma, or pancreatic.
Family history of breast cancer related to BRCA1 or BRCA2 mutations or other related genes.
Women under 60 with triple-negative breast cancer, regardless of family history.
Breast cancer diagnosis at age 45 or younger.
A history of multiple breast cancers.
Personal history of male breast cancer.

Test Methodology

BRCA1 and BRCA2 testing begins with a blood or FFPE block sample. Thermo Fisher Scientific uses Polymerase Chain Reaction (PCR) to amplify sections of the BRCA1 and BRCA2 genes. The DNA is then prepared for sequencing, which involves fragmentation, end repair, and adapter ligation. Thermo Fisher’s sequencing uses Ion Torrent next-generation sequencing (NGS) technology. Their bioinformatics tools analyze the data, identify genetic variations, and assess their implications based on established cancer risk associations.

Sample Requirements

Blood (3-5 ml in EDTA tubes)
FFPE block
Required forms: Relevant clinical information, test request form

Endometrial Receptivity Analysis (ERA)

The Endometrial Receptivity Analysis (ERA) test is a state-of-the-art diagnostic tool used in reproductive medicine to evaluate the readiness of the uterine lining for embryo implantation. Designed for women undergoing assisted reproductive treatments such as IVF, this test provides tailored information about the ideal timing for embryo transfer by analyzing the molecular characteristics of the endometrium. The ERA test aims to identify the optimal implantation window, offering potential improvements in pregnancy success rates for cases of recurrent implantation failure or unexplained infertility.

ERA testing is recommended for:

Women with recurrent implantation failure despite good-quality embryos
Those with unexplained infertility where no other causes have been identified
Cases with inconsistent implantation success across IVF cycles
Women over 35, where fertility and implantation success rates decline
Personalized fertility treatments when standard protocols have not worked

The test involves collecting an endometrial biopsy during a specific phase of the menstrual cycle, extracting DNA, and analyzing it through next-generation sequencing (NGS) and bioinformatics to determine receptivity based on gene expression patterns. Required samples include endometrial tissue and relevant clinical information.

Preimplantation Genetic Testing (PGT)

Preimplantation Genetic Testing (PGT) is a cutting-edge technique in reproductive medicine that evaluates embryos created through in vitro fertilization (IVF) for genetic abnormalities before they are transferred to the uterus. This advanced testing addresses various genetic issues, including chromosomal disorders, single gene disorders, and chromosomal structural rearrangements. By analyzing embryos at the blastocyst stage, PGT allows fertility specialists to select those with the highest chance of implantation and healthy development, thereby enhancing the probability of a successful pregnancy for couples undergoing assisted reproduction.

PGT encompasses three main types:

PGT-A (Aneuploidy Screening): Detects numerical chromosomal abnormalities, such as extra or missing chromosomes, helping to identify euploid embryos with the correct number of chromosomes.
PGT-M (Monogenic Disorders): Tests embryos for specific genetic mutations linked to single gene disorders, useful when parents are known carriers of conditions like cystic fibrosis or sickle cell disease.
PGT-SR (Structural Rearrangements): Focuses on identifying embryos with balanced chromosomal arrangements when parents have chromosomal rearrangements, reducing the risk of miscarriage and genetic disorders.

The process involves biopsying embryos at the blastocyst stage, analyzing them with Next-Generation Sequencing (NGS) for chromosomal abnormalities, and selecting euploid embryos for transfer. PGT can significantly lower the risk of miscarriage, aid couples with recurrent pregnancy loss or advanced maternal age, and offer personalized fertility treatments based on detailed genetic information.

Carrier Sequencing

In today’s diverse society, genetic disorders that were once confined to specific ethnic groups are increasingly appearing across broader populations. Traditional carrier screening, which targets single disorders based on ancestry or family history, may not accurately reflect these changing frequencies. Advances in genetic analysis, such as next-generation sequencing (NGS), are now making it possible to conduct carrier screening for a wider array of disorders with greater accuracy, speed, and cost-effectiveness.
The Ion Torrent™ CarrierSeq™ Kits, designed specifically for carrier sequencing research, offer a comprehensive solution for expanded carrier screening. These kits integrate all necessary components for library preparation, template preparation, sequencing, and data analysis, enabling laboratories to perform NGS-based screening efficiently.

Key Benefits

Broad Carrier Detection: The 420-gene panel included in the CarrierSeq Kits can analyze over 28,000 non-benign ClinVar variants, including single-nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) using NGS. This broad coverage enhances the detection rates for a wide range of inherited disorders.
Improved Lab Efficiency: The kit consolidates challenging targets—such as those complicated by pseudogenes or paralogs—into a single NGS assay. This approach streamlines the process and improves overall lab efficiency.
Simplified Implementation: The end-to-end solution is optimized for ease of use, leveraging Ion AmpliSeq™ technology. The intuitive software facilitates quick data analysis and reporting, ensuring reliable and consistent results.

Rigorous Design and Performance

The CarrierSeq Kits analyze over 28,000 variants across 420 genes linked to 418 inherited disorders. The panel includes approximately 14,000 amplicons covering coding sequences and intron/exon boundaries, ensuring comprehensive SNV and CNV analysis. Expert design and algorithm development enhance CNV detection and enable robust analysis despite challenges like pseudogenes and gene paralogs.