NGS Presentation

1. NEXT
GENERATION
SEQUENCING
AYESHA ZULQARNAIN
Sequenc
ing

2. CONTENT
 INTRODUCTION TO SEQUENCING
 HISTORY OF SEQUENCING
 GENERATION OF SEQUENCING
 NEXT GENERATION SEQUENCING
 METHODOLOGY OF NGS
 ADVANCEMENT & ADAPTATION IN
NGS
 APPLICATION OF NGS
Sequencing

3. Sequencing
3
In genetics and biochemistry, sequencing means to
determine the primary structure (sometimes incorrectly
called the primary sequence) of an unbranched biopolymer.
Sequencing refers to the general laboratory technique for
determining the arrangement of nucleotides, or bases, or
amino acid in a DNA, RNA and protein molecule.

4. DNA sequencing
 Sanger sequencing
 Whole genome sequencing (WGS)
 Targeted sequencing
 Whole exome sequencing (WES)
 Amplicon sequencing
 Molecular inversion probes (mips)
 Next-generation sequencing (NGS)
RNA sequencing
 Whole transcriptome sequencing (WTS)
 Targeted gene expression with RNA-sequencing
 Ribosomal RNA depletion
Protein sequencing
 N-terminal amino acid sequencing
 C-terminal amino acid sequencing
Typesofsequencing
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5. TimelineofSequencing projects
1977 1981 1990 1995 1996 2001 2005 2008 2011 2019
Sanger Method
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Human mitochondrial
genome sequence
Complete eukaryotic
genome
Complete cell
genome Month, 20YY
Complete human
genome project
Second generation
sequencing
Human Genome
project
Research human
microbiome project
Third generation
sequencing
Third generation
microbiome project

6. NEXTGENERATION SEQUENCING
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• Next-generation sequencing (NGS) is a massively parallel sequencing. (A high-
throughput method used to determine a portion of the nucleotide sequence of an
individual's genome.) technology that offers ultra-high throughput, scalability,
and speed.
• Next-generation sequencing (NGS), also known as high-throughput sequencing
• Using NGS an entire human genome can be sequenced within a single day.

7. PrincipleOf NGS
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 The fragments of DNA or RNA are ligated with adaptors to create genomic
libraries which are sequenced in the massively parallel event.
 The final results are reassembled and analyzed to study the genome

8.  High-throughput DNA Sequencing Technique.
 Employs Micro and Nanotechnologies
 Reduce sample size.
 Low reagent cost
 less time
 Massive Parallel Sequencing
 Sequence thousands of sequences at once.
 Produce enormous amount of data.
Keyfeatures ofNextGeneration Sequencing
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9. MethodologyofNGS
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 Sample preparation
i. Isolation of DNA
ii.Fragmentation
iii.Ligation of DNA adaptors
iv.Denaturation
v. Binding to chips
Amplification
i. DNA amplification by PCR
ii.Bridge binding & bridge amplification
iii.Denaturation
Sequencing
i. Sequencing by synthesis
Data analysis
i. Compare sequency with reference DNA or template

10. Illustration ofthenext-generation sequencingprocess.
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11. Animation of
NGS
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12. FourmainDNAsequencingmethodsusedbyNGSsystems
Sequencingbysynthesis
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Pyrosequencing
Sequencingbyligation Ionsemiconductorsequencing
 The pyrosequencing method is based on the
detection of the pyrophosphate release on
nucleotide incorporation. Due this
incorporation light is emitted which is
detect by detector
 Large read lengths generation
 High reagent cost
 High error over 6+ homopolymers
 Sequencing by synthesis utilized the step
by step incorporation of fluorescent.
Sequencing by synthesis (SBS) technology
uses four fluorescently labeled
nucleotides to sequence. When a
nucleotide incorporate a specific light
emitted which detect by detector
 Increased error rate with increased read
length
 Synthesis by ligation used DNA ligase. It
used 16 8-mer oligonucleotides probes,
each one of 4 fluorescent dyes
 Used for very short read lengths
 Ion semiconductor sequencing utilized the
released of hydrogen ions during
sequencing. Sequencing conducted on a
semiconductor transistor which capable of
detecting changes in the pH of solution
 Similar to pyrophosphate
 More cost-effective and time efficient

14. ApplicationsofNGS
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 Rapidly sequencing of whole genomes
 Deeply sequence target regions to detect mutation
 Utilize RNA sequencing (RNA-Seq) to discover novel RNA variants and
splice sites, or quantify mRNAs for gene expression analysis
 Analyze epigenetic factors such as genome-wide DNA methylation and
DNA-protein interactions
 Sequence cancer samples to study rare somatic variants, tumor
subclones, and more
 Study the human microbiome
 Identify novel pathogens

15. ApplicationsofNGS
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 Higher sensitivity to detect low-frequency variants
 Lower limit of detection
 Higher capacity with sample multiplexing
 Ability to sequence hundreds to thousands of genes or gene regions
simultaneously
ChallengesinNGS
 Less cost-effective for sequencing low numbers of targets (1–20
targets)
 Time-consuming for sequencing low numbers of targets (1–20 targets)

16.  Author links open overlay panelJames M.HeatherPersonEnvelopeBenjaminChain,
M.HeatherPersonEnvelope, J., BenjaminChain, Highlights•We review the drastic changes to DNA
sequencing technology over the last 50 years.•First-generation methods enabled sequencing of
clonal DNA populations.•The second-generation massively increased throughput by parallelizing
many reactions.•Thir, & AbstractDetermining the order of nucleic acid residues in biological
samples is an integral component of a wide variety of research applications. Over the last
fifty years large numbers of researchers have applied themselves to the production of
technique. (2015, November 10). The sequence of sequencers: The history of sequencing DNA.
Genomics. Retrieved January 14, 2023, from
https://www.sciencedirect.com/science/article/pii/S0888754315300410
 Behjati, S., & Tarpey, P. S. (2013, December). What is next generation sequencing? Archives of
disease in childhood. Education and practice edition. Retrieved January 14, 2023, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841808/
 Biogene blog. (n.d.). Retrieved January 14, 2023, from https://www.creative-
biogene.com/blog/index.php/2016/11/01/brief-introduction-on-three-generations-of-genome-
sequencing-technology/
References
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17.  DNA sequencing fact sheet. Genome.gov. (n.d.). Retrieved January 14, 2023, from
https://www.genome.gov/about-genomics/fact-sheets/DNA-Sequencing-Fact-Sheet
 Embl-Ebi. (n.d.). What is next generation DNA sequencing? What is Next Generation DNA
Sequencing? | Functional genomics II. Retrieved January 14, 2023, from
https://www.ebi.ac.uk/training/online/courses/functional-genomics-ii-common-technologies-and-
data-analysis-methods/next-generation-sequencing/
 Illumina sequencing technology. (n.d.). Retrieved January 14, 2023, from
https://www.illumina.com/documents/products/techspotlights/techspotlight_sequencing.pdf
 Integrated DNA Technologies. (2021, August 23). Types of DNA and RNA sequencing. Integrated
DNA Technologies. Retrieved January 14, 2023, from
https://sg.idtdna.com/pages/community/blog/post/let-s-talk-about-sequencing
References
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18.  Kchouk, M., Gibrat, J.-F., & Elloumi, M. (-1, November 30). Walsh Medical Media: Journals:
Open Access Journals. Biology and Medicine. Retrieved January 14, 2023, from
https://www.walshmedicalmedia.com/open-access/generations-of-sequencing-technologies-from-
first-to-next-generation-24326.html
 Mignardi, M., & Nilsson, M. (2014, April 28). Fourth-generation sequencing in the cell and the
clinic - genome medicine. BioMed Central. Retrieved January 14, 2023, from
https://genomemedicine.biomedcentral.com/articles/10.1186/gm548
 What is next-generation sequencing?: Thermo Fisher Scientific - US. What is Next-Generation
Sequencing? | Thermo Fisher Scientific - US. (n.d.). Retrieved January 14, 2023, from
https://www.thermofisher.com/pk/en/home/life-science/sequencing/sequencing-learning-
center/next-generation-sequencing-information/ngs-basics/what-is-next-generation-
sequencing.html
 Wikimedia Foundation. (2022, November 29). Sequencing. Wikipedia. Retrieved January 14, 2023,
from
https://en.wikipedia.org/wiki/Sequencing#:~:text=In%20genetics%20and%20biochemistry%2C%20seque
ncing,sequence)%20of%20an%20unbranched%20biopolymer.
References
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19. THANKYOU
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