A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time. DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene.

1. DNA Sequencing (more)
• DNA sequencing is a laboratory technique used to
determine the exact sequence of bases (A, C, G, and
T) in a DNA molecule.
• The DNA base sequence carries the information a
cell needs to assemble protein and RNA molecules.
• The information is important to investigate the
functions of genes.
• The technology was made faster and less expensive
as a part of the Human Genome Project.
Genome.gov
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2. DNA Sequencing
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3. The Human Genome Project
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4. DNA sequencing
• De novo genome sequencing
and assembly
chromosome
l
eukaryotic
viral
prokaryotic
2000 – draft human genome
sequence
2003 – completed (kind of)
Ensembl genomes:
- 69 higher animals +
other model animals
- 55 insects and lower
metazoans
- 39 plants
- 563 fungi
- Over 200 protist
species and subspecies
- Over 20 000 bacteria
species and subspecies

5. Genomics
• Area of genetics that concerns the sequencing and
analysis of an organism’s genetic information
• DNA sequencing + bioinformatics => sequence,
assemble and analyze the function and structure of
genomes (the complete set of DNA within a single cell
of an organism)
Bacterial genome
Human genome

6. The Human Genome Project
• First draft genome of human in 2001, final 2004
• Estimated costs $3 billion, time 13 years
• Used Sanger Sequencing
Today:
Illumina: 1 week, 9500$
Exome: 6 weeks*, $1000
Towards 1000$ genome
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7. • Takes advantage of miniaturization to engage
in massively parallel analysis
– Essentially carrying out millions of sequencing
reactions simultaneously in each of 10 million tiny
wells
• Sophisticated computer analysis of huge
amounts of information allows “assembly" of a
given sequence.
Next (second) Generation Sequencing

8. NGS
• These techniques could be used to
– deal with similar problems than microarrays,
– but also with many other.
• They raised the promise of personalized medicine
• NGS technologies have been on the market only since 2004
• Have now largely replaced Sanger sequencing technologies (owing
to the ultra-high-throughput production/hundreds gigabases) .
• The advent of high-throughput sequencing technologies has
initiated the ‘personal genome sequencing’ era for both normal and
cancer genomes .
• Large-scale international projects such as the 1000 Genomes
Project and the International Cancer Genome Consortium
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9. NGS Technologies/Platforms
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10. NGS Technologies/Platforms
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11. Sequencing Principles
• Sequencing by Synthesis
– Sanger/Dideoxy chain termination (Life Technologies, Applied
Biosystems)
– Pyrosequencing (Roche/454)
– Reversible terminator (Illumina )
– Ion torrent (Life Technologies)
– Zero Mode Waveguide (Pacific Biosciences) 3rd GS
• Sequencing by Oligo Ligation Detection
– SOLiD (Applied Biosystems)
• Direct reading of DNA sequence
– Nanopore sequencing 3rd GS
– Electron microscope 3rd GS

12. NGS, General Procedure
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13. NGS Process in different Platforms
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14. Roche Sequencing Process
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• DNA Library Preparation

15. Nanopore sequencing (direct reading)3rd generation
sequencing

16. NGS Application
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NGS
Whole Genome
Seq
Exome Seq
RNA-seq
Epigenetic
Metagenomics
Resequencing
Gene
Regulation

17. NGS Application

18. • Ancient DNA
• DNA mixtures from diverse ecosystems, metagenomics
• Resequencing previously published reference strains.
• Identification of all mutations in an organism
• Errors in published literature
• Expand the number of available genomes.
• Comparative studies
• Deciphering cell’s transcripts at sequence level
without knowledge of the genome sequence
• Sequencing extremely large genomes, crop plants
• Detection of cancer specific alleles avoiding traditional cloning
• Chip-seq: interactions protein-DNA
• Epigenomics
• Detecting ncRNA
• Genetic human variation : SNP, CNV (diseases)

19. • Degraded state of the sample  mitDNA sequencing
• Nuclear genomes of ancient remains: cave bear, mommoth, Neanderthal (106 bp )
Problems: contamination modern humans and coisolation bacterial DNA

20. • Key part in regulating gene
expression
• Chip: technique to study DNA-
protein interaccions
• Recently genome-wide ChIP-
based studies of DNA-protein
interactions
• Readout of ChIP-derived DNA
sequences onto NGS platforms
• Insights into transcription
factor/histone binding sites in
the human genome
• Enhance our understanding of the
gene expression in the context of
specific environmental stimuli

21. • ncRNA presence in genome difficult to predict by
computational methods with high certainty because the
evolutionary diversity
• Detecting expression level changes that correlate with
changes in environmental factors, with disease onset and
progression, complex disease set or severity
• Enhance the annotation of sequenced genomes (impact of
mutations more interpretable)

22. • Extreme example: multiplexing
the amplification of 10 000
human exons using primers
from a programmable
microarray and sequencing
them using NGS.

23. • Characterizing the biodiversity found on Earth
• The growing number of sequenced genomes enables us to interpret partial
sequences obtained by direct sampling of specif environmental niches.
• Examples: ocean, acid mine site, soil, coral reefs, human microbiome which may
vary according to the health status of the individual

24. • Common variants have not yet
completly explained complex disease
genetics rare alleles also contribute
• Also structural variants, large and
small insertions and deletions
• Accelerating biomedical research

25. • Enable of genome-wide patterns of
methylation and how this patterns
change through the course of an
organism’s development.
• Enhanced potential to combine the
results of different experiments,
correlative analyses of genome-wide
methylation, histone binding patterns
and gene expression, for example.

26. Microarray technique
Concept, methodology and applications

27. DNA microarray is an innovative technology that
facilitates the analysis of the expression of
thousands of genes simultaneously.
The utilization of this methodology, which is
rapidly evolving, requires a combination of
expertise from the biological, mathematical and
statistical sciences.

28. • The rapid advance of genome-scale sequencing has
driven the development of methods to exploit the
information encoded by such genes and to define
their participation in physiological and disease
processes.
• Microarray technology seems likely to become a
standard tool for both molecular biology research and
clinical diagnostics. This could be achieved by the
systematic survey of RNA, DNA and even protein
variation.

29. • The main advantage of this high throughput
method is that it allows generating information
of thousands of genes in a single experiment.
• DNA microarray is thus the latest in a line of
techniques to exploit a potent feature of the
DNA duplex: the sequence complimentarity of
the two strands.

30. Types of Microarrays
-Expression Arrays
-Protein microarrays (Proteomics)
-Resequencing arrays
-CGH arrays- Comparative genomic
hybridization
-SNPArrays
-Antibody Arrays
-Exon arrays-Alternative splice variant
detection
-Tissue Arrays

31. Building the chip
Arrayed Library
(96 or 384-well plates of
bacterial glycerol stocks)
PCR amplification
Directly from colonies with
SP6-T7 primers in 96-well
plates
Consolidate into
384-well plates
Spot as microarray
on glass slides

32. Spotted cDNA and Oligo
Glass Arrays:
Involves two dyes on the
same slide
• Red dye-Cy5
• Green dye-Cy3
• Control and
experimental cDNA on
same chip

33. Expression profiling with cDNA microarrays
cDNA “A”
Cy5 labeled
cDNA “B”
Cy3 labeled
Hybridization Scanning
Laser 1 Laser 2
+
Analysis Image Capture

35. Image analysis of cDNA array

36. Electroniconically Addressable Microarrays
Motorola- eSensor Chip
Nanogen- Nanochip

38. Tissue Arrays
Slide based “spotted” tissues (not really)

39. Assembling
Tissue Arrays
Coring of
embedded
paraffin tissues
and plugging
or inserting
into
new paraffin
block
Sectioning and
deposition
onto a slide