Introduction to DNA sequencing

1. Introduction to DNA Sequencing
Introductory Molecular Techniques Workshop
17th- 21st August 2015
Gezahegn Girma

2. What is DNA?
• DNA stands for:
D: Deoxyribose
N: Nucleic
A: Acid
• It is the carrier of genetic
information/blue print of
life.
• In simple terms, DNA
contains the instructions for
making proteins within the
cell

3. The DNA molecule
- a double helix with two
strands.
- The backbone of the
molecule is alternating
phosphates and
deoxyribose sugar
- The teeth are
nitrogenous bases.

4. Nucleotides
One deoxyribose together
with its phosphate and
base make a nucleotide.
• Cytosine C
• Thymine T
• Adenine A
• Guanine G
Organic base
deoxyribose
PO4
The bases always pair up in the
same way

5. The central Dogma of molecular Biology
- is an explanation of the
flow of genetic information
within a biological system.

6. Protein Synthesis: Translation
•There are 43 = 64 codons
for the 20 amino acids. G
A
A
Arginine

7. What is DNA sequencing?
• is the process of
determining the precise
order of nucleotides/the
four bases—A, G, C, and
T—in a strand of DNA.

8. Types of DNA sequencing
– eg. Sequencing of
PSY gene, rbcL
1. Targeted gene sequencing

9. 2. Reduced representation sequencing (GBS, RAD)
(PstI,(6bp) (CTGCA^G))

10. 3. Whole genome sequencing
- Is a laboratory process that determines the complete
DNA sequence of an organism's genome at a single
time.

11. 1. Genetics (Variation at fundamental level/DNA)
Why DNA sequencing?

12. eg. trait- marker association study, GWAS
2. Plant and Animal improvement

13. 3. Species identification (DNA barcoding)
-Uses short genomic region that is
universally present in target lineages and
has sufficient seq. variation to
discriminate among spp

14. 4. Metagenomics
• is the study of genetic
material recovered
directly from
environmental samples.
• Some Microbes are
unculturable!!

15. - the study of cellular and
physiological trait
variations that are not
caused by changes in
the DNA sequence
- Methylation status??
- Methylated DNA
sequencing
5. Epigenetics

16. 6. Comparative Genomics
• Comparison of whole
genome sequences of
different species.
• Provides a highly
detailed view of how
organisms are related to
each other at the
genetic level

17. - important tool to
guide therapeutic
intervention
- predicting disease
susceptibility and
drug response
7. Diagnostic (medicine, virology),

18. 8. Biotechnology,
9. Forensic biology,
.
.
.

19. DNA sequencing technologies
Maxam-Gilbert , Sanger
1970’s
454-Illumina- SOLiD
Pacific Bioscience, Heliocs and
Ion Torrent
Single molecule sequencing

20. Sanger Sequencing
Normal dNTP
(Extends DNA strand)
ddNTP
(terminates synthesis)
- Template DNA
- Primer
- DNA polymerase
- Normal dNTP
- + Small amount of ddNTP

21. i. PAGE (1970s)
Run four reactions
(ddTTP,ddATP,ddGTP,ddCTP)
650-1000bp
each reaction has many molecules each
one incorporating its respective ddNTP
and stopping at a different length.
Image from Wikipedia

22. ii. Advances in Sanger (1990s)
- One Vs Four rxn
- Labeled ddNTP with
different fluorescent colors
- Color as a function of
position
Image from Wikipedia
FluorescenceRadioactivity

23. Capillary electrophoresis
Þ16- 96 reads/run

24. Read Length
Þ Initially read length of Sanger sequencing
rarely > 25 bp.
Þ Read length is now approximately 800 to
1000 bp.
Þ First 15 to 40 bases have poor quality, and it
deteriorates again after 700-900 bases.

25. Next/New generation sequencing
Roche -454
pyrosequencing
Solexa/Illumina-
Genome Analyzer
Reversible
terminator
chemistry
SOLiD
Ligation based
extension

26. • One can sequence
hundreds of millions of
short sequences (35bp-
100bp) in a single run.
Ilumina AB/life tech
Helicos
454
(Roche)
Capillary
based (ABI)
~50 ~ 300 ~700
Mb
Kb
Gb
High throughput DNA sequencing

27. Template/library preparation methods
DNA
Add adaptors
on to the end
Attachment to solid surface
like bead that has little
sequence complementary to
this adaptor
Sequence and
analysis
Shear

28. Annealing single stranded
DNA to beads that has
complementary DNA on it
Create a library
One DNA molecule
One water bubble
One bead
Emulsification
(oil water mix)
Reagents necessary to do little
PCR to clonally amplify this
single molecule
+
What is measured is the
amount of light emitted.
Amount of light emitted is
proportional to the number of
A’s, C’s, G’s and T’s
454

29. Break emulsions and
Depositing DNA beads
into the Pico titer plate
Construct single
stranded adaptor
ligated DNA
Perform
emulsion PCR
(1 bead/well)
Sequencing by synthesis:
-simultaneous sequencing of the entire genome in
hundreds of thousands of pico titer size wells

30. Pyrosequencing
Þ Pyrophosphate
signal generation
By adding single
nucleotide at a time
and looking at the light
reaction coming of on
adding specific base

31. Monochrome detection/’pyrogram’
-Light intensity ≈ no. of
inserted nts
-Homopolymer problems
Nucleotide sequence
Nucleotide added
Maxam and Gilbert Sanger 454 Illumina SOLiD

32. Solexa/Illumina
Þ sequencing by synthesis (no chain
termination)
Þ Generate up to 100Gb per run
ÞAll bases added at a time unlike 454, each
base has different Fluorescent

33. DNA with
primers that
allows to
hybridize
Cluster generation-
Flow cell
Several hundreds to
Thousand of copies
Attach to solid
surface
Image acquisition
1 32 4 5
3’ 5’
5’
G
C
T
A
Base calling
T A G C…..
G
A C
T
Laser
Detect signal
Add sequencing
reagents
Remove
unincorporated
bases
Repeat

34. -No problem with Homopolymer repeats
ÞBase by base sequencing
- Each lane can sequence 24-36 million molecules
Þ8 lanes can yield > 280 million reads
- Produces data at a rate of 5-6Gb per day

35. - Sequence by oligo ligation /detection.
- The actual base detection no longer done by the polymerase-
driven incorporation of labeled dideoxy terminators.
Fragment preparation
(DNA or RNA)
Amplification in to
Beads (using emPCR)
Randomly deposit bead clones
on to slide surface
Sequence clones by ligation
based sequencing
SOLiD Work flow
SOLiD

36. SOLiD summary
-30-50Gb/run
-50bp read lengths
-Each base in this sequencing method is read twice
-The only sequencing method based on ligation chemistry.

37. Platform summary
Amplification Sequencing Read Gbp/day Cost
$/Mb
Status
Sanger Cloning Synthesis 1Kb 0.006 ~500 standard
454 emPCR Synthesis (Multi nt) 450bp 0.5 ~20 standard
Solexa PCR on slide Synthesis (single nt) 2x100bp 25 ~0.5 standard
SOLiD emPCR Ligation 2X50bp 10 ~0.5 standard

38. Quality control
PHRED quality score (Q score) /= sequence quality metrics
Sequencing data with low Q scores
It indicates the probability that the sequencer calls a given base incorrectly by
assigning a Q score represented (ASCII) characters to a base, which is equivalent
to the probability of the number of times an incorrect base is called.
The quality value Q assigned to a base-call was defined as a property that is
logarithmically related to the base calling error probabilities (p)
Q =−10 log10 p
where p is the estimated error probability for that base-call

39. Quality Scores and Base Calling Accuracy