1. 1

2. 1
P.Gangadhara Rao
IARI Ph.D
VEGETABLE SCIENCE

4. Introduction
Sequencing (DNA sequencing) is used to
determine the order of the nucleotide bases
(ATGC) in a molecule of DNA.
(Ansorge et al., 2009)

5. Cont…
DNA sequence represents a single format onto
which a broad range of biological phenomena
can be projected for high throughput data
collection
(Stenzel et al., 2008)

6. First generation sequencing
(Robertson et al., 2007)

7. Sanger method
(Sanger et al., 1977)

8. Sanger method (Helmut Kae et al., 2011)

9. Maxam and Gilbert method
• Chemical modification in the DNA
• Here purified DNA is used
• The fragments are radioactively labeled at 5’ end
• Chemical treatment generates the nucleotide bases
• DNA is cleaved by hot piperidine at modified base
• Conc. modifying chemicals is applied to the DNA
• Generates radiolabeled DNA fragments
• Electrophoresis and visualized in X-rays films
(Ordway et al., 2007)

10. (www.molecularstation.com/...techniques/maxam-Gilbert-sequencing/2009)

11. Dye-terminator sequencing
(www.nslc.wustl.edu/elgin/genomics/tour/pdf/pcr.pdf/ 2008)

12. Capillary electrophoresis
Sequence ladder by radioactive
sequencing compared to fluorescent peaks
(www.nslc.wustl.edu/elgin/genomics/tour/pdf/pcr.pdf 2008)
Cont…

13. Amplification and clonal selection
(BraslavskyI et al., 2003)

14. Next generation sequencing
(Metzker, 2010)

15. Next (2nd
) generation sequencing
platforms
(Metzker, 2010)

16. (Nature biotechnology, 2008)

18. (http://www.454.com)

19. Roche/454’s GS FLX
Titanium
(http://www.454.com.)
454 Sequencing technology

21. (Genome Analyzer brochure, http://www.solexa.com)

22. (http://www.solexa.com)

23. 11/10/2010 Advanced Centre for
Biotechnology
23
Fluorescent Signal Detection
Laser
Energy
CCD
Electrophoresis
+
-
dye
• Laser beam generated by single argon laser
split to form dual pathway
• Split beam allows simultaneous illumination
of 16 (4) capillaries from both sides of array at
detection cell
• Fluorescence signal emitted from DNA
fragments at the detection cell are collected
on CCD camera

24. SOLiD DNA sequencing platform
(http://www.appliedbiosystems.com)

25. SOLiD DNA sequencing platform
(http://www.appliedbiosystems.com)

26. (Schuster et al., 2008)
Sequencing-by-ligation using the SOLiD DNA sequencing
platform

27. (Johnson, 2009)

28. (Schuster et al., 2008)
Sequence determination by the
SOLiD DNA sequencing platform

29. Cont…
(Johnson, 2009)

30. High-throughput shotgun
Sanger sequencing
(Nardis et al., 2011)

31. ( Nardis et al., 2011)
High-throughput shotgun Sanger sequencing

32. ( Nardis et al., 2011)

33. Shotgun sequencing with
cyclic-array methods
( Nardis et al.,
2011)

34. (Nardis et al., 2011)

35. (Nardis et al., 2011)

36. Comparison of next (2nd
) generation sequencing platforms
Platforms Lib/ template
preparation
Prospects Constrains Biological
applications
Roche/ 454’s
GS FLX
Titanium
Frag/ emPCR Longer reads
improve
mapping in the
same regions;
fast run times
High reagent
cost; high error
rate occurrence
Bacterial and
insect genome
assemblies
Illumina/
Solexa’s GA II
Frag/ solid-
phase
Currently the
most widely
used platforms
Low samples
reads
Gene discovery
in
metagenomics
Life/APG’s
SOLid 3
Frag/ emPCR Error correction
is possible
Long time runs Gene discovery
in
metagenomics
Helicos
BioSciences
HeliScope
Frag/single
molecule
Genome and
sequenced
based
applications
High error rate
occurrence
Sequenced
based methods

37. 3636

38. Shotgun Sequence of the Melon Genome
(Jordi et al., 2011)

39.  In total, more than 22 million reads from a mixture of shotgun
and 3,820 kb paired-end reads representing 17.6x of the melon
genome have been sequenced and assembled.
 The current melon assembly contents 361Mbp(80%) of
expected genome sequence of double haploid line.
 The melon genome assembly that its quality is high and has
47,837 scaffolds and 1.1 Mb obtained using pure shotgun reads.
 Sanger sequencing is more accurate but more cost whereas
Roche method is less accuracy and lower in cost.
 This studies have also been done in watermelon, cucumber.
(Jordi et al., 2011)

40. Whole genome shotgun sequencing of Brassica oleraceae and
its application to gene discovery and annotation in
Arabidopsis
Comparative studies of A. thaliana and close related
species of Brassica oleraceae has been identified and
represent as a functional sequence.
A total of 454,274 whole genome shotgun sequences
covering 283 Mb of the estimated 650Mb Brassica
genome were searched against Arabidopsis genome
(Ayele et al., 2011)

41. Cont..
229,735 conserved genes 167,375 genes intersected with
Arabidopsis gene model
After removal of the matching known proteins, the
genome was chained together as a comprising portions of
same functional units.
(Ayele et al., 2011)

42. This resulted in 27,347 chains of 15,686 genes were distinct
from existing gene expression
By Rapid amplification method of cDNA ends was used to
obtain potential full length transcripts region of 58 genes
This resulting sequence led to creation of 21 gene models and
17 new overlapping genes against Arabidopsis genome
Collectively this resulted data sets suggests that several
thousands of new Brassica genes has to be identified
(Ayele et al., 2011)

43. Comparison between Brassica sp and Arabidopsis thaliana

44. Genome of the Cucumber, Cucumis sativus L.
Nature genetics 41, 1275- 1281(2009)
Published online: 1 November 2009 I doi:10.1038/ng.475
The genome sequence of Cucumis sativus var. sativus L.,
assembled using a novel combination of traditional Sanger and
next- generation Illumina GA sequencing technologies to
obtain 72.2- fold genome coverage.
The absence of recent whole-genome duplication, alone with
the presence of few tandem duplications, explains the small
number of genes in the cucumber
(Nature genetics, 2009)

45. Cont…
The sequenced cucumber genome affords
insight into traits such as its sex expression,
disease resistance, biosynthesis of
cucurbitacin and fresh green odor.
Identified 686 gene clusters related to phloem
functions and also evolution of vascular
system
(Nature genetics, 2009)

47.  Potato Genome Sequencing Consortium (PGSC) aims to
elucidate the complete genome sequence of potato.
 This strategy is being complemented by whole genome shotgun
sequencing approaches using both 454 GS FLX and illumina
GA2 platforms for BAC-by-BAC sequencing.
 This allows the parallel sequencing of several BACs in one
sequencing of several BACs tagged with Multiplex Identifiers
increasing the sequencing the speed and reducing costs
(Visser et al., 2009)

48. The strategy for this is to combine anchored BAC- by-BAC based
sequenced data with the random short read sequence data that can
be generated by the Illumina GA2 and the ROCHE GS FLX
platforms.
56 BAC were sequenced by traditional Sanger's method.
Among them 24 BAC contiguous DNA stretch sizes have been
predicted by pulsed field gel electrophoresis (PFGE).
Then 8 BACs were sequenced followed by 48 BACs in two
consecutive runs, using two reaction chambers and was predicted
using PFGE (Visser et al., 2009)

50. Category Examples of applications
Complete genome resequencing Comprehensive polymorphism and
mutation discovery in individual
human genomes
Reduced representation
sequencing
Large scale polymorphism
discovery (tomato)
Targeted genomic resequencing Targeted polymorphism and
mutation discovery (tomato)
Paired end sequencing Discovery of inherited and
acquired structural variation
Metagenomic sequencing Discovery of infectious and
commensal flora (potato)
Applications of next (2nd
) generation sequencing

51. Category Examples of applications
Transcriptome sequencing Quantification of gene expression
and alternative splicing; transcript
annotation; discovery of
transcribed SNPs or somatic
mutations (cucumber, tomato)
Small RNA sequencing Micro RNA profiling (tomato)
Chromatin immunoprecipitation-
sequencing (ChIP Seq)
Genome-wide mapping of protein-
DNA interactions (cucumber)
Nuclease fragmentation and
sequencing
Nucleosome positioning
Molecular barcoding Multiplex sequencing of samples
from multiple individuals
(Brassica sp)

52. Third generation sequencing technologies
• SBS technologies in which single molecules of DNA
polymerase are observed as they synthesize a single
molecule of DNA
• Nanopore-sequencing technologies- molecules of DNA
are threaded through a nanopore and individual bases are
detected
• Direct imaging sequencing of individual DNA
molecules using advanced microscopy techniques
• Hence the above technologies are currently in pipeline
(http://www.pacificbiosciences.com/oxfordjournals.org)

53. Conclusion
Future generation sequencing
 Requires only a smaller space area to work
 To determine the length of the DNA, gel
electrophoresis is not needed
 Hundreds of reactions can be run at any one time
 It requires short time to sequence a DNA molecule
 It is cost effective

54. Overall view of the 1st
and 2nd
generation
sequencing