Griffith and the Transforming Principle

A. The Concept
The experiments of Griffith and Avery, MacLeod and McCarty are closely related.
Griffith developed the concept of the transforming principle. The prinicple was
able to transform a non-pathogenic bacteria into a pathogenic strain. Changing
phenotype is one of the characteristics of the hereditary material. Griffith called
the factor that changed the phenotype the tranforming principle. Avery, McCarty,
and MacLeod performed a series of experiments that demonstrated the hereditary
materials was DNA..

Live Live Heat-killed Live Heat-killed

Type IIR Type IIIS Type IIIS Type IIR Type IIIS
+

Mouse lives. Autopsy result: Mouse lives. Autopsy result:

Live Type IIIS cells Live Type IIIS cells

Fred Griffith's experiments provided the Griffith was able to show that if you heat kill a
experimental platform for Avery, McCarty, and Type IIIS strain and injected it into the mouse, the
MacLeold to prove the DNA was the genetic mouse lived. But if you mixed the heat-killed type
material. He worked with the pathogenic bacteria IIIS material with live type IIR bacteria, the mouse
Streptococcus pneumoniae that is lethal to mice. would die. Furthermore, the autopsy showed
But not all types of the bacteria all lethal: type R is that the mouse became infected with the Type IIIS
non-lethal, whereas type S is lethal. In addition, strain. These meant that some material from the
there are type II an III strains of the bacteria. Each Type IIIS strain was taken up by the Type IIR
of these can be either R or S. So a Type IIIS strain strain to convert it into the Type IIIS strain. Griffith
is lethal, whereas a type IIR is non-lethal. termed the material the transforming principle.

One feature of the genetic material is its ability to control phenotype. In Griffith's experiment, the bacterial
strains have several phenotypes. The R types are not only non-lethal, and they have a rough (R) appearance
on a blood agar plate. The S type are distinct from the R type: they are lethal and have a smooth morphology
on the plates. The S types have a polysaccharide capsule that is lacking in the R types. Each capsule type
is distinguished using antibodies; the type II capsule is antigenically distinct from the type III. The trans-
formation from type II to type III and the conversion of type R to S are each distinct phenotypic changes.
Therefore if the chemical nature of the transforming principle could be determined, then we would know
the nature of the genetic material. Avery, MacLeod and McCarty found the answer.

Figure 1. The experiment of Griffith that demonstrated the concept of the

transforming principle.
 Avery, MacLeod and McCarty:
DNA Is The Genetic Material

A. The Concept
Avery, MacLeod and McCarty extended the work of Griffith. They used his
system, but rather than working with the mice they only studied the bacterial
phenotypes relative to the material from the dead type IIIS. They performed
careful analysis and proved that DNA, and not protein or RNA, was the genetic
material.

Type IIR Heat-killed Type IIR

Cells IIIS Cells Antibody Enzyme
Type IIR
cells
+
Type IIIS
cells
+ + +
Type IIIS
cells
+ + + Protease
Type IIIS
cells
+ + + RNase

No
+ + + DNase cells

No
+ cells

Rather than work with mice, Avery, MacLeod and McCarty used the phenotype of the
Streptococcus pneumoniae cells expressed on blood agar. To ensure, a few potentially live
cells did not escapte the heat treatment, they also precipitated those cells out of culture using
an antibody to the type IIR cells. Finally, they included an enzyme treatment of the the material
from the heat-killed cells. Each of these enzyme destroyed either proteins (protease), RNA
(RNase), or DNA (DNase). These are the three main components of the heat-killed cells. As you
can see above, the only treatement that prevented the conversion of the type IIR cells to type IIIS
was DNase. This demonstrated conclusively that DNA was the transforming principle and the
heredity chemical of life.

Figure 2. The experiment of Avery, MacLeod and McCarty that demonstrated that
DNA was the genetic material.
 DNA Structure
A. The Concept
DNA has a regular structure. It's orientation, width, width between nucleotides,
length and number of nucleotides per helical turn is constant. All of these
features were described by Watson and Crick. Adenine is always opposite
thymine, and cytosine is always oppostie guanine. The two strands are held to-
gether by hydrogen bonds: two bonds between adeninine and thymine and three
bonds between guanine and cytosine.

3' 5'
3' 5'
.
G .. C

.
C .. G
.
T . A
.
T .A
Minor
One helical turn = Groove
0.34 nm .
3.4 nm = G .. C
between .
nucleotides C .. G
10 nucleotides A .. T
Major
Groove
.
G .. C
.
T .A

This figure describes the general features 5' 3'

of B DNA, the most common structure found 5' 3'
within a cell. Other forms of DNA also exist.
All forms have unique features. These are: 2.0 nm Anti-parallel
orientation
Helix Nucleotides Helix
Form Direction per turn Diameter
A Right 11 2.3 nm
B Right 10 2.0 nm
Z Left 12 1.8 nm

Figure 3. The structure of common DNA molecules.

 Deoxyribonucleotide Structure

A. The Concept
DNA is a string of deoxyribonucleotides. These consist of three different
components. These are the dexoyribose sugar, a phosphate group, and
a nitrogen base. Variation in the nitrogen base composition distingushes each
of the four deoxyribonucleotides.
Basic deoxyribonucleotide components

O O O
Nitrogen
γ β α 5'
O P O P O P O CH 2 Base
O
O O O C4' 1' C
Phosphate 3' 2'
Group H C C H

OH H
The basic building block is the deoxyribose
Sugar
sugar. This sugar is distinguished because it Moiety
contains a hydrogen (H) atom at the number
2' carbon. Normal ribose has a hydorxyl (-OH) Nitrogen Bases
group at this position.
Purines Pyrimidines
Attached to the 5' carbon is a triphosphate H
group. This group is important because in a N
*O
DNA chain it undergoes a reaction with the
N
C
H
* C
4 CH 2
3' OH group to produce polydeoxynucleotide. 6

HC8
7 C5 1 N
* * HN 3 5 C

The final feature of the molecule is a nitrogen

9 C4 2 CH C2 6 CH
N 3 1

base. These are attached to the 1' carbon. Four N O N

bases are possilbe. Two pyrimidines (thymine and
cytosine) and two purines (adenine and guanine). Adenine Thymine
The double stranded DNA molecule is held together H
by hyrodgen bonds. Pairing involves specific atoms O
* N
in each base. Adenine pairs with the thymine, and
guanine pairs with cytosine. These pairings and N
C
6
* H
C
4

the atoms involved are shown to the right. HC 8

7 C 5 1 NH
* H
*N 3 5 CH
9 C4 2 C C2 6 CH
N 3
N 1

* *
You have probaly heard of ATP, the energy molecule. N O N
H
It is the deoxyribonucleotide to which adenine is
attached. This molecule serves two very important Guanine Cytosine
functions in biological organisms.

Figure 4. The structure of deoxyribonucleotides and base pairing among N bases.

 A Single Strand Molecule of DNA

A. The Concept
Each strand of the double-stranded DNA molecule has the same basic structure.
It is a series of series of deoxyribonucleotides linked together by phophodiester
bonds.

5' end
O O O Nitrogen
γ β α 5'
O P O P O P O CH 2 O Base

O O O C4' 1' C
3' 2'
H C C H

O H
Nitrogen
α 5'
O P O CH 2 Base
O
O C4' 1' C
3' 2'
H C C H

O H
Phosphodiester Nitrogen
α 5'
O P O CH 2 Base
Bonds O
O C4' 1' C
3' 2'
H C C H

OH H
DNA is a polynucleotide. It consists of a series of deoxyribonucleotides
that are joined by phosphodiester bonds. This bond joins the a phosphate
group to the 3' carbon of the deoxyribose sugar.
3' end
Each strand is complementary to the opposite strand. If one strand has
an adenine at a position, its anti-parallele strand would have a thymine at the
the corresponding position. Likewise, guanine and cytosine would be
complementary.

Fig. 5. The single strand structure of DNA.

 Making a Phosphodiester Bond/
Growing the DNA Chain

A. The Concept
The addition of a new nucleotide to a DNA molcule creates a phosphodiester
bond. This requires the DNA chain that is being elongated and a deoxyribo-
nucleotide.

5' end
O O O Nitrogen
γ β α 5'
O P O P O P O CH 2 O Base

O O O C4' 1' C 5' end

3' 2' O O O Nitrogen
H C C H γ β α 5'
O P O P O P O CH 2 O Base
O H
Nitrogen O O O C4' 1' C
α 5'
O P O CH 2 Base 3' 2'
O H C C H
O C4' 1' C
O H
H C
3' 2'
C H Nitrogen
α 5'
O P O CH 2 Base
O
O H
Nitrogen O C4' 1' C
α 5'
O P O CH 2 Base 3' 2'
O H C C H
O C4' 1' C
O H
H C
3' 2'
C H Nitrogen
α 5'
O P O CH 2 Base
O
OH H
O C4' 1' C
3' end H C
3' 2'
C H

+ O
O
α
H

P O CH 2 O
5'
Nitrogen
Base

O C4' 1' C
O O O Nitrogen 3' 2'
γ β α 5'
Base H C C H
O P O P O P O CH 2 O
OH H
O O O C4' 1' C O O
H C
3' 2'
C H
γ
O P O P O
β
3' end
OH H O O
Phosphodiester bonds are formed when a new
dideoxynucleotide is added to a growing DNA molecule.
(Pyrophosphate)
During the reaction, a condensation reaction occurs between
the α phosphate of the nucleotide and the hyroxyl group attached
to the 3' carbon. This reaction is performed by the enzyme DNA polymerase.
This is also an energy requiring reaction. The energy is provided by the
breaking of the high-energy phophate bond in the nucleotide. This results in
the release of a pyrophosphate molecule.

Figure 6. The formation of the phosphodiester bond that grows the DNA chain.
 Steps of DNA Replication
(Part 1)
A. The Concept
DNA replication is an essential biological process. It's primary function is to produce
new DNA for cell division. The process has several distinct steps that are important
to understand. The factors that are absolute requirements for DNA replication to
begin are a free 3'-OH group and a DNA template. A RNA primer provides the
free 3'-OH group. The DNA to be replicated serves as the template. It is important
to remember that all DNA replication proceeds in the 5'-3' direction.
1. The replication fork is formed; RNA primer added. 2. DNA is replicated by the 5'-3' synthesis function of DNA
polymerase using the leading strand in a continuous manner.
Replication Fork

Continuous
5' 3' 5' 3'
Replication
5' OH-3' 5'
3' 5' 3' 5'

RNA Primer
Leading
OH-3'
Strand

3. An error occurs during DNA replication. 4. The DNA replication error is removed by 3'-5' exonuclease
function of DNA polymerase.

5' Replication 3' 5' 3'

Replication
5' Error 5'
3' 5' 3'
Error Removed 5'

TA A C TT OH-3'

ATT G AT ATT G AT

Notes on E. coli replication:

DNA Polymerase I and III. Pol III is the primary replicase enzyme that performs the elongation of the
DNA strand. It adds nucleotides first to the RNA primer and then grows the chain by creating the
phosphodiester bonds. It also has a 3'-5' proofreading (exonulcease) function that removes incorreclty
incorporated nucleotides. DNA Pol I also has the 5'-3' replicase function, but it is primarily used to fill
the gaps in the replicated DNA that occur when the RNA primer is removed. This enzyme also has a
5'-3' exonuclease function that is used to remove the RNA primer.

Figure 7. The steps of DNA replication.

 Steps of DNA Replication
(Part 2)

5. The DNA replication error is corrected. 6. Meanwhile, Okazaki fragments are synthesized using the
lagging strand in a discontinuous manner while the leadng strand
is completed simultaneously.

5' 3'OH
Lagging
Strand
Okazaki Fragments
(Discontinuous replication)
5' Replication 3' 5' 3'
OH-3' 5'
5' Error Corrected 5'
3' 5' 3' 5'

3 '
-OH
TA A C TA
Leading
Strand
AT T G AT
7. The RNA primers are removed by 5'-3 exonuclease function 8.. Replication is completed by the filling in the gaps by DNA
of DNA polymerase. polymerae and DNA ligase.

3'OH
5'
5'

5' 3' 5' 3'

OH-3' 3' 5'
5' OH-3' 5' 3'
3' 5' 3' 5'

Notes on replication:

Okazaki fragments: Both prokaryotic and eukaryotic DNA replication proceed in the 5'-3' direction. This
poses a problem because the replication fork only moves in that one direction. The problem relates to what
is called the lagging strand. It must be replicated in a direction that is opposite of the direction of the
replication fork. This problem was solved by the discovery of Okazaki fragments (named after the person
who discovered the process). In contrast to the leading strand, in which DNA is replicated as a single
molecule in a continuous manner, DNA is replicated in a disocontinuous manner on the lagging strand.
Each fragment requires a RNA primer, and DNA Pol III in E. coli makes short stretches of DNA. These
fragments are then stitched together when the primer is removed, and the strands are completed by the
action of DNA Pol I and ligase.

Figure 7 (cont.). The steps of DNA replication.

 Chain Termination Sequencing:
the Sanger Technique
A. The Concept
DNA sequencing is the most important technique of genomics. By collecting the sequence
of genes and genomes we begin to understand the raw material of phenotype devel-
opment. The most common DNA sequencing technique is called chain termination
sequencing or the Sanger technique (named after the person who created it). It is called
chain termination because the incorporation of a dideoxynucleotide terminates the
replication process because this nucleotide lacks the required 3'-OH group.

a. A dideoxynucleotide b. The reaction reagents

O O O
Nitrogen DNA template
γ β α 5'
O P O P O P O CH 2
O
Base sequencing primer
O O O C4' 1' C dNTPs
Phosphate
H C
3' 2'
C H ddNTPs (low concentration)
Group
Note: neither the DNA polymerase
H H 2' or 3'carbon
Sugar
has an OH group salts
Moiety

c. The sequencing reaction result: fragments that differ

by one nucleotide in length
Template A T T C G G A T C C T T A A
Primer 5' T A A G C C T A G G A A T T - H 3'
5' T A A G C C T A G G A A T - H 3'
5' T A A G C C T A G G A A - H 3'
5' T A A G C C T A G G A - H 3'
5' T A A G C C T A G G - H 3'
5' T A A G C C T A G - H 3'
5' T A A G C C T A - H 3'
5' T A A G C C T - H 3'
5' T A A G C C - H 3'
When a dideoxynucleotide is inserted, the
5' T A A G C - H 3' DNA replication process terminates because
dideoxynucleotides do not have the necessary
5' T A A G - H 3'
free 3' hydroxyl group required for the addition of
5' T A A - H 3' additional nucleotides. This results in fragments that
differ by one nucleotide in length.
5' T A - H 3'
5' T - H 3'

Figure 8. The chain termination (Sanger) DNA sequencing technique.

 Gel-based Detection of DNA Sequences
A. The concept
Four DNA sequencing reactions are performed. Each contains only one of the four
dideoxynucleotides. Each reaction is added to a single lane on the gel. Since one of
the dNTPs is radioactive, the gel in which the fragments are separated, can be used
to expose an x-ray film and read the sequence.

a. The sequencing products b. The sequencing gel

Reaction with ddGTP G A T C
A T T C G G A T C C T T A A

5' T A A G C C T A G G - H 3'
3'
5' T A A G C C T A G - H 3' T
5' T A A G - H 3'
T
Reaction with ddATP A
A
A T T C G G A T C C T T A A
5' T A A G C C T A G G A A - H 3'
5' T A A G C C T A G G A - H 3' G
G
5' T A A G C C T A - H 3'
5' T A A - H 3'
5' T A - H 3' G
Reaction with ddTTP T
A T T C G G A T C C T T A A C
5' T A A G C C T A G G A A T T - H 3'
C
5' T A A G C C T A G G A A T - H 3'
5' T A A G C C T - H 3' G
5' T - H 3'
A
Reaction with ddCTP A
A T T C G G A T C C T T A A
T
5' T A A G C C - H 3' 5'
5' T A A G C - H 3' The sequencing reactions are separated on a
polyacrylamide gel. This gel separates the
fragments based on size. The shorter fragments
run further, the longer fragments run a shorter
distance. This allows the scientists to read the
sequence in the 5'-3' direction going from the
bottom to the top of the gel.

Figure 9. Gel-based detection of DNA sequencing products.

 Fluorescent Sequencing and
Laser Detection
A. The Concept
Rather than using four different reactions, each with a single dideoxynucleotide, the
advent of fluorescently labeled dideoxynucleotide enabled 1) the sequencing reaction to
be performed in a single tube, and 2) the fragment could be detected by laser technology.
Originally, the products were separated in a polyacrylamide gel prior to laser detection.
The introduction of capillary electrophoresis, coupled with laser detection enabled
the detection of up to 96 products at a time.

B. The Reaction Products and Analysis

A T T C G G A T C C T T A A
5' T A A G C C T A G G A A T T - H 3'
5' T A A G C C T A G G A A T - H 3'
5' T A A G C C T A G G A A - H 3' Sequencing products are
loaded on to a capillary
5' T A A G C C T A G G A - H 3' electrophoresis unit and
5' T A A G C C T A G G - H 3' separated by size.

5' T A A G C C T A G - H 3'
5' T A A G C C T A - H 3'
5' T A A G C C T - H 3'
5' T A A G C C - H 3'
5' T A A G C - H 3'
5' T A A G - H 3'
5' T A A - H 3' Laser detection and software analysis
5' T A - H 3' detects the first shortest fragment as ending
in a T (thymine). All fragments are detected
5' T - H 3' and intrepreted in the same manner.

The Sequence Chromatogram

5' T A A G C C T A G G A A T T 3'

Figure 10. The fluorescent sequencing and laser detectiion process of DNA sequencing.
DNA Autoradiogram: Original Output Data from Sequencing
Output from Automated DNA Sequencer
Cheaper Sequencing: The Wave of the Future

Based on:
Bentley (2006) Current Opinion in Genetics and Development

What are the types of sequencing projects?

• Whole genome
o Collecting the full sequence of a genome
 Draft
• 90-95% coverage
 Finished
• 98-99% coverage
• Resequencing
o Targeted sequencing for localized regions
 Discover localized changes
 Requires reference sequence

Human Genome Project

• $1 billion to complete
o 99% of euchromatic region of the genome
o 99.995% accuracy
2004 Costs
• Sanger technique
• $10 million for individual
What if it cost:
• $100,000 (100X cost reduction)
o Sequence 24-48 humans
o Discover genome-wide SNP
• $10,000 (1000X cost reduction)
o Sequence about 500 humans
 Discover evolution patterns
• Genes undergoing natural selection can be
discovered
• Presumably key genes that “make humans
humans”
o Deep sequencing of specific disease gene
 Discover key mutations associated with onset of
disease
• $1000 (10,000X cost reduction)
o Personal genome characterization
What Was Needed for All New Approaches

Reducing Cost
• How: Parallel sequencing
o Large number of sequencing reactions occurring
simultaneously
 Requires high density reactions matrix
• Many reactions in a small space
• Miniaturization of reaction unit or space
o Reduce reagent cost
 Accomplished when above factors achieved

Throughput
• Many reactions occurring simultaneously
o Current Sanger macrocapillary system
 96-384 reactions per machine
 Sequencing centers have 30-60 machines
 New approaches must have significantly greater
throughput

Sequence Accuracy Must Be Maintained

• Sanger procedure highly accurate
o Well understood Phred scores reported
 New systems will require quantifiable accuracy scores
Completeness
• Read length issue
o Sanger technology with capillary detection
o 500-700 nt
 Allows for assembly into
• Contigs
• Supercontigs
• Emerging technologies
o Length requirement
o Must be long enough to align accurately
 25-100 nt read length
• Sufficient for resequencing with a reference
genome
• Whole genome sequencing
o 100 nt (or longer) needed for smaller genomes
o Other advances needed for larger genomes
How Large Scale Sequencing Has Changed Over Time
From a Centers Perspective

Then: DOE/JGI Sanger Sequencing Equipment Room

Recently: DOE/JGI Illumina GAII Equipment Room

Now: DOE/JGI Illumina HiSEQ Equipment Room

Now: DOE/JGI PacBIO Equipment Room

 454 Sequencing Reaction Principles
Emulsion with
sequencing template
Fibreoptic plate

C T C
G A A
T A G

Step 1: add dATP

and other reagents
C T C
G A A
TA 1X light A G
emitted

Step 2: add dtTP

and other reagents
C T C
G AT A
TA AT 2X light G
emitted

Step 3: add dCTP

and other reagents

C T C
GC AT A
TA 1X light AT GC 1X light
emitted emitted
 The Sequencing Technology
Enhance your sequencing process —
from genome to sequence in record time

Generate tens of millions of bases per run with sample-library fragments, and contain a streptavidin
the straightforward workflow of the Genome binding site for sample purification. Low molecular
Sequencer 20 System (Figures 3-6). weight DNA is used without fragmentation and
sample preparation begins with adaptor ligation.
DNA Library Preparation The A and B adaptors can also be added during
Sample preparation is dependent on the type of PCR by using the appropriate primers (provided in
starting material used. The preparation process GS emPCR Kit II (Amplicon A, Paired End) and
comprises a series of enzymatic steps to produce GS emPCR Kit III (Amplicon B). The sstDNA
single-stranded template DNA (sstDNA) incorpo- library produced at the end of this preparation
rating primer and binding adaptors. For example, step is assessed for its quality, and the optimal
genomic DNA (gDNA) is fractionated into smaller amount (DNA copies per bead) needed for emPCR
fragments (300-800 base pairs) that are subsequently is determined by a titration run.
Process polished (blunted). Short Adaptors (A and B) are
Steps
then ligated onto the ends of the fragments. These
adaptors provide priming sequences for both
amplification (emPCR) and sequencing of the

DNA Library Preparation and Titration emPCR Sequencing

4.5 hours 10.5 hours 8 hours 5.5 hours

gDNA sstDNA library

■ Genome fragmented by nebulization
■ No cloning; no colony picking
■ sstDNA library created with adaptors. The adaptors are used as primers, and for binding to beads.
■ A/B fragments selected using streptavidin-biotin purification

Figure 3: DNA library preparation with the Genome Sequencer 20 System.

6|
emPCR Amplification
The sstDNA library is immobilized onto specially captured within its own microreactor for PCR
designed DNA Capture Beads. Each bead carries a amplification. Amplification is performed in bulk,
single sstDNA library fragment. The bead-bound resulting in bead-immobilized, clonally amplified
library is emulsified with amplification reagents in DNA fragments that are specific to each bead.
a water-in-oil mixture. Each bead is separately

DNA Library Preparation and Titration emPCR Sequencing

4.5 hours 10.5 hours 8 hours 5.5 hours Process

Steps

Anneal sstDNA Emulsify beads and Clonal amplification Break microreactors,

to an excess of PCR reagents in occurs inside enrich for DNA-
DNA Capture water-in-oil microreactors positive beads
Beads microreactors

sstDNA library Clonally-amplified sstDNA attached to bead

(millions of copies per bead)

Figure 4: Overview of emulsion-based clonal amplification (emPCR) with the Genome Sequencer 20 System.

|7
 The Sequencing Technology
Enhance your sequencing process —
from genome to sequence in record time

Sequencing-by-Synthesis If a nucleotide complementary to the template

Sequencing starts with the preparation of a strand is flowed into a well, the polymerase
PicoTiterPlate device; during this step, a extends the existing DNA strand by adding
combination of beads, sequencing enzymes, nucleotide(s). Addition of one (or more)
and an sstDNA library is deposited into the nucleotide(s) results in a reaction that generates
wells of the device. The bead-deposition a chemiluminescent signal that is recorded by
process maximizes the number of wells that the CCD camera in the Genome Sequencer 20
contain an individual sstDNA library bead. Instrument. The signal strength is proportional
to the number of nucleotides incorporated in a
The loaded PicoTiterPlate device is placed into single nucleotide flow.
the Genome Sequencer 20 Instrument. The
fluidics subsystem flows sequencing reagents
Process (containing buffers and nucleotides) across
Steps the wells of the plate. Each sequencing cycle
consists of flowing individual nucleotides in a
fixed order (TACG) across the PicoTiterPlate
device. During the nucleotide flow, each of the
hundreds of thousands of beads with millions
of copies of DNA is sequenced in parallel.

DNA Library Preparation and Titration emPCR Sequencing

4.5 hours 10.5 hours 8 hours 5.5 hours

■ Well diameter: average of 44 µm

■ A single clonally amplified sstDNA

bead is deposited per well

■ 200,000 reads obtained in parallel

on large-format PicoTiterPlate device

Amplified sstDNA library beads Quality reads

Figure 5: Deposition of DNA beads into the PicoTiterPlate device.

8|
 DNA Library Preparation and Titration emPCR Sequencing

4.5 hours 10.5 hours 8 hours 5.5 hours

Signal image
■ Bases (TACG) are
sequentially flowed
(42 times)

Polymerase ■ Chemiluminescent
signal generation

■ Signal processing
to determine base Process
APS sequence and Steps
Annealed quality score
PPI primer

Sulfurylase
ATP
Luciferin
Luciferase
DNA Capture Bead
containing millions
of copies of a single
clonally amplified Light + Oxy Luciferin
fragment

Amplified sstDNA library beads Quality reads

Figure 6: Sequencing reaction of the Genome Sequencer 20 System.

|9
 The Genome Sequencer 20 Instrument
Perform ultra-high-throughput DNA sequencing

The Genome Sequencer 20 System revolutionizes Optics Fluidics

DNA sequencing, delivering sequence data in a —— Subsystem———————————— ————————-—Subsystem ——
massively parallel fashion.
The Genome Sequencer 20 System includes:
■ Instrument and accessories Sipper
————————— Manifold
■ Status
Reagents and consumables for library Indicator ———————
construction, amplification, and sequencing Pumps,
CCD Valves, and
■ Camera
Analysis software for resequencing, de novo Face ———————————
——————————————– Debubblers

assembly, and amplicon sequencing. PicoTiter- —————

Reagents
———————————— Cassette
Plate
Cartridge
The instrument (Figure 7) is the centerpiece of the —————
Genome Sequencer 20 System. It comprises both
Computer ——————————— Keyboard
optics and fluidics subsystems, which are controlled Subsystem and Mouse
by a computer subsystem. in drawer
—————
Genome The fluidics subsystem consists of a reagents
Sequencer cassette, a sipper manifold, pumps, valves, and Figure 7: Open view of the Genome Sequencer 20
20 System Instrument.
debubblers. It ensures accurate reagent dispensing
and flows the sequencing reagents across the wells Instrument space requirement: 30 in (77 cm) Wide x 36 in
(92 cm) Deep x 69 in (176 cm) High (cart and instrument).
of the PicoTiterPlate device.
The optics subsystem includes a CCD camera,
Position of PicoTiterPlate
which captures the light signal resulting from the Device in the
Instrument
sequencing reaction (Figure 8). Peristaltic
Pump

CCD
Sipper Camera
Manifold

Reagents Cassette PicoTiterPlate Device

Figure 8: Expanded view of the Genome Sequencer 20

Instrument components. Arrows represent reagent flow.

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Illumina Sequencing by Synthesis Technology
Illumina Sequencers Over Time: Today’s Workhorse

Illumina GAII [Maximum (Max) output: 25 gigabases (Gb)]

Illumina HiSeq 2500 (Max output; 500 Gb; Rapid Run Mode: 150 Gb)
 Today’s Illumina Models
(Mostly chemistry and reads per flow cell differences)

Illumina NextSeq (Max output: 120 gigabases)

Illumina HiSeq X10 (Max output: 1.8 Tb) GENOMES ONLY

Illumina HiSEQ 4000 (Max output: 1.5 Tb) Most other sequencing

Illumina NovaSeq (Next Generation; 2017 release; Max output: 1.5 Tb)
Single Polymerase Real Time DNA Sequencing
Developed by Pacific Biosciences
Sequences occurs at the rate of 10 nt per second

Principle

Reaction Cell

• A single DNA polymerase is immobilized on the bottom of a

reaction cell
o Reaction cell called a ZMW (Zero-mode waveguide)
• Ф29 DNA polymerase is used
o Fast single subunit enzyme.
• Each sequencing plate contains ~3000 individual cells
o Each holds only a single DNA molecule

Chemistry

• A phospholinked dNTP is used

o Each dNTP contains a different fluorophore
• During sequence
o A single labeled dNTP enters the polymerase
o dNTP held in place shortly
o Fluorescence signal is emitted in the ZMW for a short period
of time
o dNTP leaves and new dNTP enters

Detection and sequence determination

• Fluorescence signals for each ZMW collected

o Data is collected as a movie of the sequential signals
 Each individual signal is measured as a short pulse of
light
o Successive fluorescence signal data is collected
o DNA sequence of single molecule is determined by sequence
of light pulses
Images and Notes Below From:
Pacific Biosciences Technology Backgrounder (11/24/2008)

Title: Pacific Biosciences Develops Transformative DNA Sequencing

Technology: Single Molecule Real Time (SMRT) DNA Sequencing

ZMW (Zero-mode waveguide) with Ф29 DNA polymerase and DNA

template
Base-labeled dNTP

Phospho-labeled dNTP
Single Polymerase DNA Sequencing

Step 1: Fluorescent phospholinked labeled nucleotides are

introduced into the ZMW.
Step 2: The base being incorporated is held in the detection volume
for tens of milliseconds, producing a bright flash of light.
Step 3: The phosphate chain is cleaved, releasing the attached dye molecule.
Step 4-5: The process repeats.
Original Potential Advantages
Speed
• 10 nt per second

Length
• 1000-2000 nt
• This is a claim
o Not fully proven

Assembly
• Much easier to assembly longer fragments

Cost
• Company claim
o $10/human genome

2010
• Only a few of these machines have been delivered
o Advantages remain to be determined fully

2017 Results
• Seeing 30 kb reads today
o Company is increasing throughput with new machine
• Many genomes are being sequenced by this technology
o This technology solves the problem of assembling repeat
regions
o Very cost effective
 Today
• $35,000 genomes primarily based on PacBio
sequence data
 Other Devices Available Today

Illumina MiSeq Personal Sequencer (Sequencing-by-Synthesis)

Ion Torrent (pH Sequencing)

 Semi-conductor Sequencing
Ion Torrent/Proton Torrent
(Life Technologies)
Chemistry
• Similar to 454 technologies
• Template bead is in an emulsion bead that is loaded into a single well
• Sensor detects the release of a hydrogen ion as a nucleotide is
inserted into the growing polynucleotide
• Differs from 454 which detects the release of pyrophosphate
o Nucleotides sequentially washed across the chip
o If nucleotide is inserted, H+ released and pH changes
o Semi-conductor detects pH changes
o pH changes indicates number of nucleotides inserted
 no signal, that base not next in the sequence
 2x signal; two of the same base in order

Chip consists of three layers

• Microwells that hold DNA template
• Ion sensitive layer
• Ion detector
o Small pH meter
Features of the Life Technologies Instruments

Feature Ion Torrent PGM Proton Torrent

Application Small set of genes Whole large genomes
Small genomes Transcriptomes
Gene expression
Throughput 10 Mb - 1 Gb 10 Gb
Read length 35-400 bp 200 bp
# Reads passing filter 60-80 million
Sequencing run time 1.5 hr (100 bp reads) 2-4 hr
Chips • 314 (1 million wells, • Proton I (165
10 Mb) million wells; 2
• 316 (6 million wells, human exomes)
100 Mb) • Proton II (600
• 318 (11 million million wells; 1
wells, 1 Gb) human genome
ION TORRENT PROTON TORRENT
 Alternative Library Preparation Protocols

10X Genomics
Synthetic Long Read Seqeuencing

Addresses assembly problems

• Hard to link short reads (Illumina) into “long” contigs
• Longer contigs make final assembly of contigs into scaffolds easier
• Goal of 10X Genomics sequence library preparation:
o Collect reads associated with a single genomic locations and
assembly them into “artificial long reads”

Workflow
Methodology
• DNA fragment (up to ~100kb) is loaded into an emulsion bead
similar to 454 technology
• Emulsion bead created by fluidic partioning device

Emulsion micells (called GEM) contains

• Anchor bead
• DNA
• One 16bp barcode (out of ~4 million)
• Enzyme to fragment DNA??
• dNTPS
Isothermal Amplification
• Multiple fragments from a long fragment are amplified
• All contain the same barcode

GEM pooling

• All GEM are pooled

• GEMs emulsion dissolved and all barcoded fragments pooled

Sequencing and assembly

• Illumina sequencing library developed with the barcoded
fragments
• Library sequenced using Illumina technology
• Reads are portioned based on barcodes
• Contigs assembled from each barcode pool
• Contigs across barcode pools assembled into longer conting.
Assembled synthetic molecule (from 10X, Deanna Church)

• Note different colors on ends and that each original fragment is

not completely sequenced.
• Multiple barcoded fragments pooled to create overlapping contig

Variant Discovery

The two allelic regions of an individual differ by a deletion

Human Trio Analysis
 Dovetail Genomics Sequencing

from: https://dovetailgenomics.com/technology/

Hi-C linking
• Based on links between natural interactions within a chromosome
o Regions of the chromosome are associated via chromatin
o Based on principal that DNA has a 3-D confirmation in the
cell
o 3D configuration occurs because controlling elements that
regulate a gene’s expression are not always immediately
adjacent to coding region of the gene

Chicago
• An artificial linking procedure
• When used with Hi-C, the Hi-C derived relationships can be
confirmed
from: http://science.sciencemag.org/content/326/5950/289/tab-pdf

Hi-C procedure
1. Crosslink the cells using formaldehyde to stick chromosomes
together
2. Isolate “crosslinked” DNA bound with chromatin
3. Digest DNA with six-cutter restriction enzyme
4. Fill ends and add biotin to end
5. Ligate ends and pull down molecules with biotin procedure
6. Sequence pull down library using Illumina paired-end protocol

Assembly
• Long distance relationships can be used during assembly
• Distances between ends are typically >20Kb
• Data can be used in the final steps of assembly.
Illumina Paired End Sequencing
Illumina Mate Pair Library Sequencing
 General Steps That Apply To ALL
Massively Parallel DNA Sequencing Systems

1. Isolate DNA
• Care is needed to ensure the DNA is of uniform high quality

2. Fractionate DNA into appropriate size for specific sequencing system

• Length will vary depending on the read length you will be generating

3. Amplify individual DNA fragments that will be sequenced

• This could be in a reaction emulsion bead (Roche 454) or reaction matrix
(Illumina or Pacific Biological Science [PacBio])

4. Load DNA samples onto DNA sequencing matrix

• The matrix can be a solid chip with individual wells (Roche 454, PacBio) or a
chip with sequencing oligonucleotides (Illumina)

5. Perform sequencing reactions

• Varies from system to system

6. Collect DNA sequence data for each read

• Varies from system to system
Sequencing Only a Subset of the Genome

• Genes are the functional component of much of the genome

• They encode proteins that have the following functions:
o Enzymatic
o Regulatory
o Structural
• Genes only make up a portion of the genome
o Human: 3% of the length of the genome is gene sequence
 5’-UTR (untranslated regions)
 Exons
 Introns
 3’-UTR
• Many of the functional mutations are in the exon (coding) region
of the genes
• Exons are only a portion of the gene sequence
o They make up only a still smaller portion of the genome
o Humans: 1% of the length of the genome is exons

• Goal of exon sequencing

o Discover sequence changes just in the exon
o Associate the sequence change with change in function
From: NimbleGen
 Sequence Capture Protocol (NimbleGen)
1. Genomic DNA: SeqCap EZ Oligo pool is made against target
regions in the genome.
2. Library Preparation: Standard shot-gun sequencing library is
made from genomic DNA.
3. Hybridization: The sequencing library is hybridized to the SeqCap
EZ Oligo pool.
4. Bead Capture: Capture beads are used to pull down the complex
of capture oligos and genomic DNA fragments.
5. Washing: Unbound fragments are removed by washing.
6. Amplification: Enriched fragment pool is amplified by PCR.
7. Enrichment QC: The success of enrichment is measured by qPCR
at control loci.
8. Sequencing-Ready DNA: The end product is a sequencing library
enriched for target regions, ready for high throughput
sequencing.
Sequencing the Expressed Portion of the Genome

• Genes are expressed in a the following manners

o Tissue-specific (where)
o Temporal specific (when)
o Quantitatively (how much)
• Transcriptomics
o The study of gene expression
• Massively parallel sequencing has changed the study of the
transcriptome
o All the genes at a specific place or time can be accurately
quantified
• Procedure
o RNA-seq or massively parallel RNA sequencing
 Very powerful
 Can monitor expression of even rarely expressed genes
 RNA-seq procedure

1. Isolate RNA from target tissue

2. Select mRNA using poly-T primers
• Based on principle that all mRNA in eukaryotes have a poly-tail
3. Perform first and second strand cDNA (copy DNA) synthesis to
convert mRNA into cDNA
4. Prepare cDNA for sequencing by adding appropriate sequencing
adaptors
5. Sequence the cDNA pool using a massively parallel technology
6. Align reads against a reference genome and quantify
Aligning RNA-seq Data to the Reference Sequence