The slides discuss on applications of next generation sequencing

1. NEXT GENERATION
SEQUENCING
Anne Catherine - Chew Chin Lynn - Jaya Seri - Pridhi - Shelomi Karoon - Teh Wooi Wan

2. DNA Sequencing = Determination of precise order of nucleotides within a DNA molecule
Next Generation Sequencing = ➢ High throughput screening method
➢ Massive parallel sequencing during which millions of fragments of DNA
from a single sample are sequenced in unison
What does NGS do?
✓ Provides cheaper and high throughput
alternative to DNA sequencing.
✓ Facilitates discovery of genes and regulatory
elements associated with diseases.
Types of Next Generation Sequencing
1. Target sequencing
➢ Allows identification of disease causing
mutations
➢ Diagnosis of pathological conditions
2. RNA Sequencing
➢ Provides information on entire transcriptome
of a sample in a single analysis
➢ Acts as a strong alternative to the use of
microarrays in gene expression studies.
Limitations of Next Generation Sequencing
❖ Expensive
❖ Inaccurate sequencing of homopolymer regions
❖ Sequence errors.
❖ Amplification of DNA libraries may result in
differential expansion of certain genome regions
❖ Loss of epigenetic modifications during
amplification
❖ Time consuming
(Grada & Weinbrecht 2013)
P.
K.

3. METHODS OF NEXT GENERATION SEQUENCING
S.
K.
(Morozova,O and Marra,MA 2008)

4. ❑ Pyrosequencing
• A new approach which is faster and cheaper than the dideoxy method.
• In pyrosequencing, the pyrophosphate that is released after a DNA polymerase adds a dNTP to a
growing DNA strand is detected (Qiagen 2013).
❑ Sequencing by synthesis (Illumina)
• uses clonal amplification and sequencing by synthesis (SBS) chemistry to enable rapid, accurate
sequencing (Illumina 2015).
❑ Sequencing by ligation (SOLiD sequencing)
• generates hundreds of millions to billions of small sequence reads at one time.
• It has a similar principle to pyrosequencing as the amplification of fragmented DNA on an agarose
bead is repeated (Gundogdu,O and Elmi,A).
S.
K.METHODS OF NEXT GENERATION SEQUENCING

5. Figure 1 : The workflow of integrating omics data in cancer research and clinical applications.
(BioMed Central 2013)
S.
K.

6. Experiment Source of DNA (input) Brief Description
Whole -
genome
sequencing
(WGS)
gDNA •identifies individuals complete genome sequence (coding
and noncoding regions)
•includes copy number variation (repeats and indels)
•structural rearrangements (translocation )
Targeted
“exome”
sequencing
Protein encoding gDNA •identifies the sequence for all coding regions (exons)
•includes copy number variation (repeats and indels)
•structural rearrangements (translocations)
Bisulfite -
sequencing
Bisulfite - treated DNA •Identifies site of DNA methylation (genetic imprinting)
Rizzo,JM and Buck,MJ 2015
V.
T.CLINICAL APPLICATIONS OF NGS IN CANCER
TREATMENT

7. Figure 2 : A summary of the workflow for NGS sequencing in oncology.
Gagan and Van Allen 2015
V.
T.

8. 1. Algorithmic Interpretation.
➢ PHIAL
❑ Applies a heuristic method to rank alterations by clinical and biological relevance
❑ Followed by intra-sample pathway analysis to determine potentially druggable nodes.
2. Use of Databases
❑ Catalog of Somatic Mutations in Man (COSMIC) and TCGA
❑ information about therapies and prognostic information can be found at a number of locations
❑Cancer centers that have created and host these databases include MD Anderson’s Personalized
Cancer Therapy, Vanderbilt’s My Cancer Genome and the Broad Institute’s TARGET where useful
information and links to relevant primary literature can be found.
(Gagan and Van Allen 2015)
CLINICAL INTERPRETATION OF NGS DATA
C.
L.

9. 1. Sequence heterogeneity even within single primary tumour
➢ happens as DNA mutation accumulate within a tumour
➢ challenging to decide if a read of a low allele fraction represents a true mutation that exists within a
subset of tumour cells or is an artefact that should be discarded.
➢ requires increased sensitivity to detect low allelic fraction alterations in impure tumor samples that may
impact an individual patient’s care.
2. Frequent DNA Fusions
➢ Causes both ends of read to be mappable, but the whole contiguous sequence is not.
➢ Challenging in the presence of a high number of structural rearrangements.
➢ Whole-exome sequencing assays often miss these variants, and gene panels that are not designed to
cover known fusion territories will also be unable to identify these fusion products.
(Gagan and Van Allen 2015)
CHALLENGES OF USING NGS IN CANCER THERAPY
C.
L.

10. ❑ Integrating information from different modalities
❑ RNA sequencing – relative expression of mutated gene.
❑ Mass spectrometry – gives clearer picture of proteomics of cancer.
❑ Immunotherapies – gaining prevalence for cancer therapy (Melanoma)
❑ Predicts responses to immunotherapy
❑ Exome sequencing paired with mass spectrometry – determination of neoantigens.
FUTURE DIRECTIONS
A.
C.
Gagan and Van Allen 2015

11. Figure 3 : An illustration of new clinical trial designs.
Gagan and Van Allen 2015
A.
C.

12. CASE STUDY:
CLINICAL INVESTIGATION OF EGFR MUTATION DETECTION BY
PYROSEQUENCING IN LUNG CANCER PATIENTS
● Clinical specimens from 202 lung cancer patients were analyzed for EGFR mutations in
exons 18, 19, 20 and 21 using the pyrosequencing method.
Methods :
● Patient population
● DNA extraction
● EGFR mutation analysis using pyrosequencing
● Statistical analysis
EGFR-TKI: epidermal growth receptor tyrosine kinase inhibitor.
(Kim,HJ et. al. 2012)
J.
M.

13. The results and clinical responses :
➢ EGFR-tyrosine kinase inhibitors (TKIs) were compared -
➢ EGFR mutation-positive rate was 26.7% (54/202). Activating EGFR mutations were observed more
often in female (52.1 vs. 13.0%), non-smoking (47.8 vs. 15.8%) and adenocarcinoma (35.2 vs. 5.2%)
patients.
➢ On the other hand, combinations of clinicopathological factors, including female, non-smoking and
adenocarcinoma, were not identified to significantly increase the positive EGFR mutation rate
(female, 52.1%; female and non-smoker, 52.6%; female, non-smoker and adenocarcinoma, 51.9%.
Conclusion
➢ Current study indicate that EGFR mutation analysis is a highly useful method for the prediction of
response to EGFR-TKI and the use of favorable clinicopathological factors to perform this analysis is
not suitable.
➢ The present findings shows that EGFR mutations analyzed by the pyrosequencing method are well
correlated with clinicopathological parameters and that this method may be useful in the clinical
practice.
(Kim,HJ et. al. 2012)
J.
M.

14. REFERENCES
Behjati,S and Tarpey,PS 2013, ‘What is Next Generation Sequencing’, National Center for Biotechnology Information, US National Library of Medicine,
viewed 19 October 2015, <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841808/>
Gagan.J and Van Allen, M.E 2015, ‘Next- Generation Sequencing to Guide Cancer Therapy’, Genome Medicine, vol. 7, no. 80, pp. 1-10, viewed 25
October 2015, <http://www.genomemedicine.com/content/7/1/80>
Grada,A and Weinbrecht,K 2013, ‘Next Generation Sequencing: Methodology and Application’, Journal of Investigative Dermatology,Boston University
School of Medicine, Massachusetts, USA, Department of Dermatology, viewed 19 October 2015,
<http://www.nature.com/jid/journal/v133/n8/full/jid2013248a.html>
Gundogdu,O and Elmi,A n.d., ‘Next Generation Sequencing’, Genome Resource Facility, London School of Hygiene and Tropical Medicine, London,
United Kingdom, viewed 18 October 2015, <http://grf.lshtm.ac.uk/sequencing.htm>
Illumina 2015, ‘Sequencing by Synthesis (SBS) Technology’, viewed 20 October 2015, <http://www.illumina.com/technology/next-generation-
sequencing/sequencing-technology.html>
Kaksonen,A n.d., ‘DNA Sequencing’, Tampere University of Technology, Finland, viewed 19 October 2015,
<http://wiki.biomine.skelleftea.se/biomine/molecular/index_14.htm>
Kim,HJ et al 2012, ‘Clinical Investigation of EGFR Mutation Detection by Pyrosequencing in Lung Cancer Patients’, US National Library of Medicine
Bethesda MD, USA, viewed 20 October 2015, <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3525462/>
Liu, L. et al 2012, ‘Clinical applications of next generation sequencing in cancer: from panels, to exomes, to genomes’, Rare Genomics Institute, viewed
18 October 2015, <http://journal.frontiersin.org/article/10.3389/fgene.2015.00215/pdf>

15. Morozova,O and Marra,MA 2008, ‘Applications of Next- Generation Sequencing Technologies in Functional Genomics’, Genomics, Vol. 92 No.5 pp
255 - 264 viewed 19 October 2015, <http://www.sciencedirect.com/science/article/pii/S0888754308001651>
National Human Genome Institute 2015, ‘DNA Sequencing’, United States of the America, viewed 18 October 2015,
<https://www.genome.gov/10001177>
Obenrader,S 2015, ‘The Sanger Method’, Davidson College, Davidson’s Biology, Davidson, North Carolina, viewed 18 October 2015,
<http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Obenrader/sanger_method_page.htm>
Qiagen 2015, ‘Pyrosequencing Technology and Platform Overview’, viewed 20 October 2015,
<https://www.qiagen.com/my/resources/technologies/pyrosequencing-resource-center/technology-overview/>
Rizzo,JM and Buck,MJ 2015, ‘Key Principles and Clinicals Applications of Next- Generation DNA Sequencing’, American Association for Cancer
Research, Cancer Prevention Research, 2015, USA, viewed 20 October 2015, <http://cancerpreventionresearch.aacrjournals.org/content/5/7/887.full>
Shyr,D and Liu,Q 2013, ‘Next generation sequencing in cancer research and clinical application’, Biological Procedures Online, vol. 15, no. 4, pp. 2,
viewed 20 October 2015, < http://www.biomedcentral.com/content/pdf/1480-9222-15-4.pdf>
Stefan,H et al 2015, ‘Clinical Applications of Next Generation Sequencing in Cancer:From Panels, to Exomes, to Genomes’, Frontiers In Genetics,
viewed 19 October 2015, <http://journal.frontiersin.org/article/10.3389/fgene.2015.00215/full>
Upadhyay,P et al 2014, ‘Applications of Next-Generation Sequencing in Cancer’, Research and Education in Cancer, Mumbai, India, viewed 20
October 2015, <http://www.currentscience.ac.in/Volumes/107/05/0795.pdf>
Veronique,G et.al 2015, ‘Next-Generation Sequencing Approaches in Cancer: Where Have They Brought Us and Where Will They Take Us?’, Open
Access Cancer, pp.1925-1958, viewed 20 October 2015, <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586802/>

16. THANK YOU