DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. Discuss the application of next generation sequencing in cancer treatment.

1. GENES AND TISSUE
CULTURE
DNA sequencing is the process of determining the
precise order of nucleotides within a DNA molecule.
Discuss the application of next generation sequencing in
cancer treatment.
By: Chan Yanqi (0317492), Lai Woon Fui (0317553), Mandary Madiiha B (0316855), Ivy Ng (0316439), Trevor Wang (0303235), Leong Jun Yee (0317542).

2. OBJECTIVES
• To understand what is Next generation sequencing and how does it work.
• The differences between old generation sequencing and new generation
sequencing
• To investigates the applications of using next generation sequencing in the
cancer treatments.
• Future applications, challenges and recent developments of next generation
sequencing.

3. INTRODUCTION: Cancer Therapy in current context
• Cancer, in its many forms, accounts for 8.2 million deaths in 2012 (GLOBOCAN, 2012).
The rapid development of DNA sequencing technologies has driven a revolution in our
understanding of this highly complex and diverse group of diseases (Devita &
Rosenberg 2012).
• There are a handful of cancer types, such as chronic myelogenous leukemia, that feature a
single and pathognomonic DNA mutation. Working on this assumption, early methods to
explore the genomic foundations of different cancers involved targeted exploration of
specific variants and genes in a low-throughput fashion.
• The success of Human Genome Project, along with the increased affordability and
reliability of sequencing, has led to the integration of genome science into clinical practice.
The use of these data to assist in diagnosis is generally referred to as precision medicine.

4. HOWEVER,
Demand for revolutionary technologies
that deliver fast, inexpensive and accurate
genome information started to arise. This
challenge catalyzed the development of
next-generation sequencing (NGS)
technologies.
We are going to highlight the importance and
potential of complete or 100% genome
sequencing.
Next Generation Sequencing…

5. WHAT IS NEXT GENERATION
SEQUENCING?
• Commonly termed as massively parallel sequencing, it is a way of capturing a
very big amount of genomic information about a cancer.
• NGS came as a revolution in genomic research. By using NGS, a whole
human genome can be sequences in a single day as opposed to the previous
Sanger sequencing technology, that took more than 10 years to deliver the
final draft of a complete human genome (Behjati & Tarpey 2013).

6. HOW DOES NEXT GENERATION
SEQUENCING WORK?
• Most NGS technologies uses sequencing by synthesis whereby each DNA
fragment to be sequenced is bound to an array, and then DNA polymerase
adds labeled nucleotides sequentially.
• A high-resolution camera captures the signal from each nucleotide becoming
integrated and notes the spatial coordinates and time.
• The sequence at each spot can then be inferred by a computer program to
generate a contiguous DNA sequence, referred to as a read.

7. STEPS OF NEXT GENERATION
SEQUENCING.
A summary of the workflow for NGS
sequencing in oncology (Adapted from
Genome Medicine 2015)

8. BENEFITS OF USING NEXT
GENERATION SEQUENCING.
1. Samples no longer need to be handled in a different manner from standard diagnostic specimens.
2. New advances enabled increasingly complicated genomic data to be obtained from an individual’s blood
sample only (Gagan et al. 2012).
3. Genetic mutations have long since been identified by Sanger sequencing, PCR, and microarrays.
However they have limitations that don’t apply to NGS.
For example, although previous methods can detect single nucleotide variants (SNVs), they have trouble
identifying larger DNA aberrations, such as large indels and structural rearrangements, which are
common in cancer.
On the other hand, whole exome and whole- genome sequencing can provide a comprehensive view of
the DNA aberrations, genetic recombination, and other mutations. Therefore, NGS platforms serve as
a good diagnostic and prognostic tool and help clinicians identify specific characteristics in each patient,
paving the road towards personalized medicine.

9. SANGER SEQUENCING VS NEXT
GENERATION SEQUENCING.
SANGER SEQUENCING.
PROS CONS
Polymerase errors
average out (Lowest error
rate)
Can only do 1 sequence
at a time
- Only produces one
forward and one reverse
read
Long read length (~ 450
bp)
Need a lot of DNA to
start with
- High cost per base (more
expensive)
NEXT GENERATION SEQUENCING
PROS CONS
Massively parallel
- Allows millions of
fragments to be
sequenced in a single run
Fewer reads of each bases
are combined so less
accurate overall
Fast turnaround time Short read length
Cheaper – Lower cost per
sample
-

10. APPLICATIONS OF NGS
1. NGS has already been applied in the clinic for cancer diagnosis and prognosis.
• For example, by doing the sequencing of the whole genome, researchers were able to
identify a novel insertional fusion that makes a classic bcr3 PML-RARA fusion gene for
a patient suffering from acute myeloid leukemia (AML). The findings brought about
alterations in the treatment plan of that patient (Shyr & Liu 2013).
• Oncology studies on the identification of individualized mutational landscapes for
tumors from patients with metastatic colorectal cancer and malignant melanoma
(identifying informative mutations in a clinically relevant time frame)

11. APPLICATIONS OF NGS
• Used paired-end sequencing to generate a comprehensive catalog of somatic mutations
in a malignant melanoma genome.
• Sequence cancer genomes from metastasis & primary breast cancer samples to
investigate the mutational evolution of cancer cells (Lee et al. 2013).
2. By using this NGS technology, clinicians have the possibility of designing patient-
specific probes that makes use of DNA found in the patient’s blood serum to monitor the
progress of a patient’s treatment and note any signs of relapse (Shyr & Liu 2013).

12. APPLICATIONS OF NGS
3. NGS is utilized by zooming in deeply in sequence
target regions (Target sequencing).
- Target multiple candidate genes in providing a
comprehensive view of the genetic makeup of cancer
patients.
(Rizzo & Buck 2012)

13. APPLICATIONS OF NGS
4. NGS on Transcriptome sequencing (RNA sequence).
• simultaneously analyzed to provide insight into the level of gene expression,
the structure of genomic loci, and sequence variation present at loci.
- Adult granulose cell tumors (GCT) using paired end RNA sequencing and
compared their sequencing results with the transcriptomes of 11 epithelial
ovarian tumors and published sequences of human genomes.
- FOXL2 gene involved in granulose cell development
- Mutation in FOXL2 is a potential driver in the pathogenesis of adult GCTs.
(Rizzo & Buck 2012)

14. TREATMENT USING NGS
• Standard Clinical Practices in Guiding Cancer Therapy
• Personalized Treatment of Cancer in Clinical Oncology
• Pancreatic Cancer Treatment
• Non-small Lung Cancer

15. Standard Clinical Practices for Cancer Therapy
• NGS, a result of multiple technological and practical advances
• Earlier protocols require outside of typical pathology workflows to harvest/obtain
the starting material.
• Standard formalin-fixed/paraffin-embedded specimens used.
• Knowledge bases of NGS are being massed
• Protocols for analysis and NGS data interpretation
• Allow clinicians easily obtain information for patients care (Gargan & Eliezer 2015)

16. Personalized Treatment of Cancer
• Low costs, shorter turnaround time,
• Due to the ability to fully sequence large number of mutations genes of all types in
single test.
• More comprehensive analysis of variation
• Able to detect all mutation types of targets gene and even chromosomal
abnormalities. Compare with Sanger sequencing.
• DNA required less
• Small amount from tumour tissue, as not always possible to obtain large quantity
(Guan et al. 2012)

17. PANCREATIC CANCER TREATMENT
• Using somatic alterations of pancreatic cancer genomes in NGS.
• Able to identify therapeutic options for patients. (Mardis 2012)
NON SMALL LUNG CANCER.
• Exon 19 micro-deletions been revealed by NGS as the most frequent
genetic alterations affecting Epidermal Growth Factor Receptor.
• Able to response to treatment with tyrosine kinase inhibitor. (Marchetti et
al. 2012)

18. RECENT DEVELOPMENTS OF NGS
Sequencing of cell-free DNA fragments (cffDNAs)  Reducing invasive medical procedures:
 Definition : Fetal DNA circulating freely in the maternal blood circulation
 Detecting Down syndrome and other fetal aneuploidies (trisomy 13 and 18) with high sensitivity
and specificity
 E.g. - By sequencing the subpopulation of cffDNAs in a pregnant mother's bloodstream belonging to her
fetus  Next Generation Sequencing plasma-based DNA test
 Detecting solid organ transplant rejection
 E.g. - increased levels of cffDNAs from a heart transplant donor's genome found in a recipient’s
bloodstream when a transplant recipient is undergoing an acute cellular rejection  deep sequencing of
cffDNAs  to detect changes in a small fraction of that DNA population which belonged to the organ
donor.
(Rizzo & Buck 2012)

19. RECENT DEVELOPMENTS OF NGS
Microbial diversification (Preclinical application)
Used to characterize microbial populations in a culture-independent fashion,
by probing for the presence and abundance of microbe-specific DNA
sequences in both tumor and normal human cell environments. (Rizzo &
Buck 2012)

20. RECENT DEVELOPMENTS OF NGS
Forensic molecular biology
Genetic markers on the Y chromosome  Y-short tandem repeats (Y-STRs)
To unambiguously resolve the male component of DNA mixtures when a high female
background is present
To reconstruct paternal relationships between male individuals
NGS  comparing more than 10 million nucleotides of the Y chromosome between two male
individuals who shared the same ancestor 13 generations ago  four genetic differences detected
Helps solves the problem of distinguishing between mixed male samples from the same parent.
(Yang, Xie & Yan 2014)

21. CHALLENGES OF NGS
• Presence of repetitive DNA
• To differentiate rare mutations in tumour from sequencing and alignment artefacts
• Tumour complexity and heterogeneity
• Deluge of data derive from NGS might take a long time to be translated into clinical relevant
information.
• Prediction of the functional effect and the identification of disease-causal variants are still in
an early phase
• Require high-performance computing and bio-informatics support.
• Quality control and standardisation of NGS experiments and data reporting.
(Reis-Filho, 2009)

22. FUTURE STUDIES OF NGS
• Forensic studies
• To move from the current descriptive and prognostic classification systems to a
functional genomic taxonomy (cancer research)
• To improve diagnosis of cancer
• To identify molecular aberrations that render tumours exquisitely sensitive to certain
therapies, resulting in exceptional responses.
• To facilitate clinical diagnosis and personalized disease-risk profiling
• Use as a diagnostic tools for higher detection rate in early onset diseases (Shyr & Liu 2013).

23. CONCLUSION
• From this presentation, we can conclude that today’s complex genomic
research questions demands a depth of information beyond normal capacity
of traditionally DNA sequencing technology. With its unprecedented
throughput, scalability and speed, NGS enables researchers to study
biological system at a level never before possible.

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