Lesson: Sequencing by synthesis - PowerPoint Presentation

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Lesson: Sequencing by synthesis

Goals:

Review central dogma and limits of DNA

Understand history and recent advances in sequencing

Understand the process (sequencing by synthesis) used to generate data in this module

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What is DNA?

10010110

(coding)

to

as

5’

GATTACA

3

’

(DNA)

to

LIFE

2

Slide3

The Central Dogma

DNA

5’

GATTACA

3’

3

Responsible for most of the

structure

and

function

of an organism

goes to

protein

Slide4

Different ways to represent DNA

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5’-ATTAGCTAGAC-3’

3D structure

2D chemical structure

Sequence

DNA sequencing

simply means

reading the sequence of the DNA

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Very small yet very big

DNA is tiny

Each letter in the DNA sequence is less than one nanometer

Genomic information is massive

3 BILLION letters in the human genome

LIFE

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So what?

What can we do with DNA sequencing?

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Are the following scenarios “Sci-Fi” or “Reality”?

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Identify whether someone is more or less likely to commit a crime

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Sci-fi.

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Figure out the identity of ancient human remains

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Reality.

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Use preserved DNA to re-create extinct plants and animals

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Sci-fi.

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Use human DNA to create a clone with the same personality

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Sci-fi.

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Track disease by monitoring toilet waste from airplanes

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Reality.

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Next-Generation Sequencing makes these advances possible

1 human genome in 13 years

~40 sequencing institutions

$3,000,000,000 per genome

16 human genomes in 3 days

1 sequencing system

$1,000 per genome

1990-2003: Human Genome Project

“Sanger sequencing”

technology

Today“Next-generation sequencing” technology

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DNA Sequencing by Synthesis

Bioinformatics Inquiry through Sequencing

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DNA synthesis

Template strand

DNA polymerase

Primer

Nucleotides

ATGAGCTTAGCTA

TACTCG

T

A

C

T

G

G

C

A

T

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DNA synthesis

ATGAGCTTAGCTA

TACTCG

T

A

C

T

G

G

C

A

T

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DNA synthesis

ATGAGCTTAGCTA

TACTCGAATCGAT

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Sequencing By Synthesis

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Sequence DNA by observing the synthesis of a complimentary strand

MiSeq

sequencer

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INLET

DNA is attached to the surface of a flow cell

OUTLET

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DNA is fixed in place while various chemicals wash over it

The camera takes pictures of DNA synthesis while it happens

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Make identical copies

Because nucleotides are so small, they are difficult to see, even when attached to fluorescent dyes

T

he sequencer copies the sample sequence to form a large group of identical sequences.

This group is called a

clusterThe fluorescent signal from a cluster is much greater than the fluorescent signal from a single strand

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OUTLET

DNA is fixed to the surface of a flow cell

INLET

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Fluorescent Dye

A

T

G

C

When excited by a laser, fluorescent dyes emit brightly colored light

Each nucleotide is attached to a unique fluorescent dye

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Blue

 A

Red

 T

Green

 G

Yellow

 C

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Sequencing By Synthesis

K.

Voelkerding

,

et al

, Clinical Chemistry, 2009

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Fluorescent Dye

Laser excites fluorophore.

Camera captures color

Each color indicates a specific base

(support.Illumina.com)

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Tracking colors

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The sequencer uses a camera to identify the color of each nucleotide as it gets added

Write the first letter of the color you see:

R

ed

B

lue

Y

ellow or

G

reen

DNA synthesis happens fast and uncontrollable

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Blocking group controls speed

3’-ATGC-5’

5’-TA

x

C

??

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Deblocker

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Speed can now be controlled

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Without blocking groups

With blocking groups

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Sequencing by Synthesis

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Primer and polymerase attach

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1. Add nucleotide

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2. Image fluorescence

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3. Remove dye

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Sequencing by synthesis

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Sequencing by synthesis

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Sequencing by synthesis

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Sequencing by synthesis

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Sequencing by synthesis

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Sequencing by synthesis

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Complete sequence

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What the camera sees:

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Image 1

Image 2

Image 3

Image 4

Image 5

Blue

 A

Red

 T

Green

 G

Yellow

 C

G

G

A

C

T

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What the camera sees:

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Image 1

Image 2

Image 3

Image 4

Image 5

Blue

 A

Red

 T

Green

 G

Yellow

 C

G

T

C

G

A

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Modeling Activity

Everyone gets a role:

DNA Polymerase

PrimerSequence complementary of primer

LaserCameraNucleotides of the original sequenceDeblocker

Nucleotides

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Image Credits

Applications

Criminal baby: toddlerhalloweencostumes.com

Jurassic world: http://www.jurassicworld.com

/Airplane poop: http://www.wired.com/2015/08/airplane-poop-help-track-global-disease-outbreaks/

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