Conservation through Next Generation sequencing

1. AAMIR WAHAB
(IBGE)

2. DNASEQUENCING
WHYSEQUENCEDNA
PURPOSEOF SEQUENCING
DNASEQUENCINGMETHODS
APPLICATIONS
CONCLUSION

3.  Process of determining the precise order of nucleotides within a DNA molecule.
 It includes any method or technology that is used to determine the order of the four
bases—
* Adenine (A)
* Guanine (G)
* Cytosine (C)
* Thymine (T)
 Advent of rapid DNA sequencing methods has greatly accelerated biological and medical
research and discovery.

4.  All genes available for an organism are turned in for use.
 Important tool for conservation biologists
 Not just sequence of genes, but also positioning of genes and sequences
of regulatory regions
 New recombinant DNA constructs must be sequenced to verify
construction or positions of mutations
 To analyze gene structure
 Relation to gene expression
 Protein conformation

5.  Deciphering “code of life”
 Detecting mutations
 Identifying human haplotypes
 Designating polymorphisms

6.  Historically there are two main methods of DNA
sequencing
1. Maxam and Gilbert method
2. Sanger method
 The advanced technique used nowadays is;
Next-Gen Sequencing
 Modern sequencing equipment still uses the principles
of the Sanger technique.

7.  A. M. Maxam and W.Gilbert-1975
 Chemical Sequencing
 Treatment of DNA with certain
Chemicals  DNA cuts into
Fragments  Monitoring of
sequences Walter Gilbert , 1932

8.  Most common approach used for DNA
sequencing .
 Invented by Frederick Sanger - 1977
 Nobel prize - 1980
 Also termed as Chain Termination or
Dideoxy method
Frederick Sanger, 1918

9.  NGS - High-throughput , multiparallel, rapid DNA sequencing
 Third generation – single molecule, real time, reduced chemistry
 Basic NGS principles – synthesis, ligation
 Basic workflow:
 SAMPLE - FRAGMENTATION - LIBRARY PREP - SEQ RUN - DATA
ANALYSIS

10.  NGS analysis may also help to better estimate relatedness and inbreeding
 Coefficients among individuals in wild populations.
 Estimate of inbreeding coefficients
 Potential to decrease this large variance
 New methods are being developed with improved results.

11.  Facilitating the analysis of several hundred of Giga-bases per day, or at the same
time thousands of SNPs
 A main application of NGS technology is the complete characterization of the entire
genome of a particular species.
 Analyzing the main model organisms (yeast, Escherichia coli, Drosophila,
Arabidopsis, mouse) and the human genome,
 Increasing number of genome sequences is currently being performed.

12.  Popular Platforms :
 Roche 454 - Pyrosequencing
 illumina Sequencing
 Newer Platforms:
 Ion Torrent

13. 1. DNA fragmentations and adaptor ligation.
2. DNA fragments are added to an oil mixture containing millions of beads.
3. Emulsion PCR results in multiple copies of the fragment.
4. Beads are deposited on plate wells ready for sequencing.

14. Fragmentation Adaptor ligation
Emulsion PCR amplification:
Bead deposition onto PicoTiter Plate (PTP):

15.  Continuous and cost effective for complete genomic sequencing
 Widely applicable
 Accurate and flexible
 Parallel process with automation
 Does not require labelled primers and nucleotides
 Don’t need Gel Electrophoresis

16.  Many crop plant genomes are characterized and a whole lot of potentially
important genetic data is collected through the use of NGS. The genome of
Hordeum Vulgare (Barley) comprises more than 5 Gb of DNA sequence of
which more than 2% is termed to be useful. So to avoid excessive
sequencing of non-informative, repetitive DNA the Pyrosequencing is
used, benefiting with time, cost and accuracy.
 Wild and landrace barley have been genotyped
 Genetic data is then used in the fields such as population genetics,
conservation genetics and molecular ecology.
 Several Arabidopsis lines have been sequenced (Arabidopsis thaliana,
Arabidopsis lyrata and others)
 whole-genome re-sequencing in crop species like Oryaza sativa (Rice) and
Zea Mays (Maize) are also determined.
Kilian B et al., 2012

17. Angeloni et al., 2012

18. 
 Protein -DNA
interaction
 Methylation analysis
Microbial
diversity
 FORENSICS
 MEDICINES
 AGRICULTURE
GENOME
 RESEQUENCING
 ANCIENT DNA
 mRNA Expression
and Discovery
 Alternative Splicing

19.  Methylation analysis
 Mutation analysis
 Microbial identification

20.  Pyrosequencing generates highly reproducible quantification of
methylation frequencies at individual consecutive CG sites.
 Pyrosequencing can detect and quantify even small changes in
methylation levels.
 The inherent quality controlling afforded by the sequence in context of
results.
 Built-in bisulfite treatment controls, eliminate manual estimation of
non-converted DNA levels
 Prevent false-positive methylation detection, ensuring reliable results.

21.  Pyrosequencing has been used to correlate DNA methylation with
tumor type and gene expression.
 Measure cellular response for the treatment with demethylating
agents.
 Assess changes in methylation state in relation to tumorigenesis and
exposure to environmental toxins.

22. Quantification of methylation at a single CG site in the p16 gene (Humans).
Methylation at 7 independent CG sites (highlighted in blue) is quantified in a single Pyrosequencing
CRomagosaetal.,
2011

23.  Pyrosequencing ensures precision and accuracy when performing highly sensitive
mutational analysis and quantifying alleles in mixed cell populations.
 Data are presented in a sequence context which serves as a built-in quality control.
 Reliable detection and quantification of sequence variation, down to at least the 5%
mutation level
 Characterization of contiguous and multivariable mutations
 Discovery of unknown mutations

24. Pyrogram of a DNA sequence featuring an insertion-deletion mutation (ATCTGCCC) and a somatic
mutation involving a single base pair substitution C vs T.
The histogram portray the number of nucleotides incorporated at each nucleotide distribution
CRomagosaetal.,
2011

25.  Pyrosequencing by synthesizing new copies of the DNA template,
provide unambiguous information.
 Pyrosequencing allows the identification of a large number of species
using a single conserved primer
 DNA extracted from multiple microbe species can be sequenced in the
same Pyrosequencing run,
 Depending on assay design, the sequence can be used to discriminate
microbial species, types and strains, or detect genetic mutations
 Eubacteria Profiling and Identification In stomachs of Mongolian gerbils
with or without Helicobacter Pylori Sun et al., 2003

26.  The impressive advances of sequencing technologies in recent years have
enabled a range of new applications, including population genetics based
on the complete genomic sequences of a large number of individuals and
sequencing of the complete genomes of highly contaminated samples of
ancient DNA from humans and other Species.
 Decreased sequencing cost and miniaturized equipment, it will become
possible for any experimental research group to perform whole genome
sequencing of both large and small genomes on a bench-top DNA
sequencer. DNA sequencing is likely to become an easily accessible
routine method, like the PCR technique is today.
 Pyrosequencing method has already shown evidence of high accuracy in
DNA sequencing and analysis of polymorphic DNA fragments in many
clinical and research settings

27.  Mardis, E. R. A decade’s perspective on DNA sequencing technology. Nature (2011) 470:198 -
203
 Metzker, M.L. Sequencing technologies – the next generation. Nature Review Genetics(2010)
11:31 – 46
 N. J. Loman, C. Constantinidou, Jacqueline Z. M. Chan, M. Halachev, M. Sergeant, C. W. Penn,
E. R. Robinson & M. J. Pallen. High-throughput bacterial genome sequencing: an
embarrassment of choice, a world of opportunity. Nature Review Microbiology 10, 599-606.
 https://icmb.utexas.edu/images/ICMB/Facilities/Pyrosequencing-and-its-applications.pdf
 www.ijlpr.com/admin/php/uploads/67_pdf.pdf