Grp#3

WHAT IS NEXT GENERATION
SEQUENCING?
PRESENTED BY:
GROUP # 03
OUTLINES
 INTRODUCTION
 HISTORICAL CONTEXT
 HOW NGS WORK
 TYPES OF NGS
 Application of NGS
 Advantages of NGS
 Challenges of NGS
 Conclusion
 Refrences
What is next generation sequencing?
 Next-generation sequencing (NGS) is a

massively parallel sequencing
technology that offers ultra-high
throughput, scalability, and speed.
 The technology is used to determine the
order of nucleotides in entire genomes
or targeted regions of DNA or RNA.
NGS has revolutionized the biological
sciences, allowing labs to perform a
wide variety of applications and study
biological systems at a level never
before possible.
History of sequencing
 1869 – Discovery of DNA
 1909 – Chemical characterisation
 1953 – Structure of DNA solved
 1977 – First genome (ФX174) Sequencing by
 synthesis (Sanger) - Sequencing by degradation
 (Maxam- Gilbert)
 1986 – First automated sequencing machine 1990
 – Human Genome Project started
 1992 – First “sequencing factory” at TIGR
 1995 – First bacterial genome – H. influenzae (1.8 Mb) 1998 –
 First animal genome – C. elegans (97 Mb)
 2003 – Completion of Human Genome Project (3 Gb)
 – 13 years, $2.7 bn
 2005 – First “next-generation” sequencing instrument
 2013 – 10,000 genome sequences in NCBI database
How NDS work?
 NGS follows a series of key steps to sequence DNA or RNA efficiently and accurately:
 1-Sample Preparation
 DNA/RNA Extraction:Genetic material (DNA or RNA) is extracted from a sample like blood, tissue, or
 saliva.
 Fragmentation:The extracted DNA is broken into small, manageable pieces (200-600 base pairs).
 End repair and adapter ligation:
 Special adapters (short DNA sequences) are attached to the ends of each fragment.Adapters help attach
 the DNA to the sequencing platform and allow identification (barcoding).
 2-Library Preparation
 A collection of prepared DNA fragments with adapters is called a library.
 Barcoding: If multiple samples are sequenced together, each sample is given a unique barcode to
 differentiate them during data analysis.
 SequencingThe prepared DNA fragments are loaded into an NGS platform. Different technologies use
 unique sequencing approaches:
 3-Illumina Sequencing (Most Common)
 DNA fragments attach to a surface (flow cell).Fragments are amplified (copied) through bridge
 amplification to create clusters of identical DNA.Fluorescently labeled nucleotides (A, T, G, C) are
added
 step by step.A camera records the fluorescent signal as each nucleotide is added, determining the
 sequence.
 Ion Torrent Sequencing
 Detects the release of hydrogen ions when a nucleotide is added to DNA, measuring pH changes.
 Nanopore SequencingDNA is passed through a nanopore (tiny protein channel).Changes in electrical
 current are measured to identify nucleotides.
 Data Analysis
 Raw Data: The sequencing machine generates raw data in the form of millions of short sequences
 (reads).
TYPES OF NGS
1. Whole genome shortgun sequencing

 Sequences the entire genome (coding and non-coding regions). Uses: Identifying
mutations, evolutionary studies, and personalized medicine.
2. Whole Exome Sequencing (WES)
 Focuses on sequencing only protein-coding regions (exons) of the genome, which make up
~1-2%. Uses: Identifying disease-causing mutations efficiently and cost-effectively.
3-Targeted Sequencing Sequences
 specific genes or regions of interest. Uses: Cancer gene panels, inherited disease testing,
and focused research studies.
 4-RNA Sequencing (RNA-Seq)
 Analyzes RNA to study gene expression and transcriptomes. Uses:
Understanding how genes are turned on/off and studying disease
mechanisms.
 5-Epigenomic Sequencing:
 Studies chemical modifications of DNA (e.g., methylation) without
altering the sequence. Uses: Epigenetic changes in cancer,
development, and aging.
 6-Single-Cell Sequencing
 Sequences genetic material at the level of individual cells. Uses:
Studying cell-to-cell variations in cancer, immunology, and
developmental biology.
 7-Metagenomic Sequencing
 Sequences genetic material from environmental samples (e.g., soil,
water, human microbiome). Uses: Identifying microbial communities,
disease-causing pathogens, and environmental studies.
Application of NGS
 1. Disease Diagnosis: Detects genetic mutations in inherited disorders.

 2. Cancer Genomics: Identifies tumor mutations for personalized treatments.
 3. Precision Medicine: Develops individualized therapies based on genetic profiles.
 4. Infectious Diseases: Tracks pathogens (e.g., COVID-19) and drug resistance.
 5. Agriculture: Improves crop yield, pest resistance, and livestock breeding.
 6. Microbiome Studies: Analyzes microbial communities in humans and ecosystems.
 7. Drug Development: Identifies genetic targets for new medicines.
 8. Reproductive Health: Assesses embryos for genetic abnormalities.
 9. Evolutionary Biology: Studies genetic relationships and ancient DNA.
 10. Epigenetics: Explores DNA modifications affecting gene expression.
Advantages of NGS
 1. High-Throughput:
 Simultaneously sequences millions to billions of DNA or RNA fragments.
 2. Fast Results:
 Complete genomes can be sequenced within hours or days.
 3. Cost-Effective:
 Costs have dropped significantly, enabling affordable large-scale sequencing.
 4. Precision and Sensitivity:
 Detects rare mutations, low-frequency variants, and subtle genetic changes.
 5. Versatility:
 Works for DNA, RNA, epigenomics, metagenomics, and more.
 6. Scalable:
 Suitable for single-gene studies or entire genome projects.
 7. Automation:
 Automated processes reduce manual errors and improve efficiency.
 8. Multiplexing:
 Allows multiple samples to be sequenced in a single run using barcoding.
 9. Wide Applications:
 Used in research, diagnostics, agriculture, drug discovery, and microbiome studies.
 10. Innovative Discovery:
 Identifies novel genes, biomarkers, and pathways for advancing science and medicine.
Challenges of NGS
 1. High Initial Costs:

 NGS platforms and instruments, like Illumina or PacBio, are expensive to purchase.
 2. Data Storage and Management:
 NGS generates massive datasets requiring significant storage and computing resources.
 3. Complex Data Analysis:
 Requires skilled bioinformaticians and advanced software for processing and interpreting
data.
 4. Error Rates:
 Errors can occur, especially in repetitive or GC-rich regions of the genome.
 5. Ethical and Privacy Concerns:
 Managing and protecting sensitive genetic data raises ethical and privacy issues.
 6. High Running Costs:
 Consumables like reagents and flow cells can be expensive for large-scale sequencing
projects.
 7. Turnaround Time for Analysis:
 While sequencing itself is fast, data analysis can take considerable time.
 8. Interpretation Challenges:
 Understanding the clinical or biological significance of detected variants is complex.
 9. Uneven Coverage:
 Some regions of the genome may have low sequencing coverage, affecting results.
 10. Accessibility:
 High costs and infrastructure needs make NGS less accessible in low-resource settings.
Conclusion
 1. Revolutionary Impact:
 NGS has transformed the fields of genomics, transcriptomics, and epigenetics with its high-throughput, speed, and
cost-efficiency.
 2. Wide Applications:
 From personalized medicine to agriculture, microbiome studies, and disease diagnostics, NGS is a cornerstone
technology across disciplines.
 3. Precision Medicine Leader:
 NGS enables tailored treatments by uncovering genetic variations, paving the way for personalized healthcare.
 4. Challenges Exist:
 While it faces issues like high costs, data complexity, and privacy concerns, advancements in technology and
accessibility are addressing these barriers.
 5. Future Potential:
 Continuous innovations such as single-cell sequencing and nanopore technology promise even greater precision and
broader applications.
 6. Key Takeaway:
 NGS is not just a tool but a gateway to understanding life at the molecular level, driving discoveries that will shape
science and medicine for decades to come.
References
 1. Books and Journals:

 Mardis, E. R. (2008). Next-generation DNA sequencing methods. Annual Review of Genomics and
 Human Genetics, 9,
 387–402.
 Goodwin, S., McPherson, J. D., & McCombie, W. R. (2016). Coming of age: Ten years of next-
 generation sequencing technologies.
 Nature Reviews Genetics, 17(6), 333–351.
 2. Web Resources:
 National Human Genome Research Institute (NHGRI): Next-Generation Sequencing Fact Sheet
 https://www.genome.gov/
 Illumina: Introduction to Next-Generation Sequencing (NGS)
 https://www.illumina.com/
 3. Scientific Articles:
 Koboldt, D. C., Steinberg, K. M., Larson, D. E., Wilson, R. K., & Mardis, E. R. (2013). The next-
 generation sequencing revolution and
 its impact on genomics. Cell, 155(1), 27–3 8.
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WHAT IS NEXT GENERATION

 Next-generation sequencing (NGS) is a

1. Whole genome shortgun sequencing

 1. Disease Diagnosis: Detects genetic mutations in inherited disorders.

 1. High Initial Costs:

 1. Books and Journals:

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