Created by Commercial Training and Enablement

3/18/23

Sequencing Technology

For Research Use Only. Not for use in diagnostic procedures.

Confidential. Do not distribute.
© 2021 Illumina, Inc. All rights reserved.
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What is DNA?

• DNA = Deoxyribonucleic Acid
• It is the carrier of genetic information
• The overall DNA structure is composed of (2) strands of DNA
• Composed of 4 bases:
• A binds with T
• C binds with G A T

• The order of the bases on a stranded DNA is called “Sequence” C G

• The order of all the bases in an organism's DNA is called “Genome”

• The human genome contains about 3.3 billion DNA base pairs Genome 3.3 Billion DNA letters

• Every cell in our body contains the entire human DNA sequence
• How were different organs created?
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What is a Gene?

• Small sections of DNA that contain instructions to operate a part of our body are called “Gene”
• The human body contains over 20,000 genes
• Genes tell the cell how to function and what trades to express
Gene
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What is a Protein?

• DNA is the carrier of the organism’s genetic information; it does not carry out the cell’s function
• Information from gene must be decoded into proteins
• Proteins determine cell type and function

Codes for
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What is Gene Expression?

• The process the cell uses to create protein from the genes in our DNA is called “Gene Expression”
• DNA codes for RNA
• Process of copying the DNA into RNA is called “Transcription”
• RNA codes for Protein
• RNA tells the cell how to make proteins in a process called “Translation”.

DNA RNA Protein

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What is a Chromosome?

• If all of the human’s DNA was stretched out and lined up end-to-end, it would total more than 2 Meter or 6 Feet
• To fit all the DNA inside the cell nucleus, DNA coils itself into a tightly packed structure called “Chromosomes”
• Human have 46 chromosomes arranged in 23 pairs
• Chromosomes from parents are passed down at random, which is one
source of genetic variability
• Sometimes (1) nucleotide might be incorporated instead of another called
“SNPs” (single nucleotide polymorphism)
• This genetic variation is what the scientists are using to study human
health
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Why are SNPs important for us?

• Some SNPs are important in the study of human health

 Used to personalized medicine
 Show the risk of developing particular diseases
 Used to track the inherited diseases of genes within families
 Used in forensics for DNA fingerprinting
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Illumina Sequencing Workflow

• Introduction to Sequencin
g by Synthesis (SBS):
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Illumina Sequencing Workflow

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Illumina Sequencing Workflow

Step 1: Library Preparation

For clustering: For sequencing: For mixing samples:

Libraries must have P5 and Rd1 and Rd2 Sequencing Libraries must have a unique
P7 binding regions on either Primer regions are used index or barcode sequence
end of a library to initiate sequencing
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Illumina Sequencing Workflow

Step 1: Library Preparation

• Library Preparation is performed on standard laboratory equipment and not on the Illumina sequencing system

• The objective of this step is to manipulate DNA  from double stranded DNA to single stranded DNA

Adapter PCR
Fragmentation Denature
Ligation Amplification

• Since the human • Add adapter • Each fragment is • DNA fragments is

DNA is 3 billion amplified separated into
base pairs long, it single-stranded
has to be cut up fragments
into smaller pieces
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Illumina Sequencing Workflow

Step 1: Library Preparation
 Created by Commercial Training and Enablement

Illumina Sequencing Workflow

 Created by Commercial Training and Enablement

Illumina Sequencing Workflow

Step 2: Cluster Generation

• The objective of cluster generation is to put the library on the

Flow Cell
Bridge
• What is a flow cell? amplificatio
• A thick glass slide with channels or lanes which are randomly n
coated with a lawn of Oligos (synthetic DNA) complementary
to library adapters
Bind single DNA
• Cluster generation occurs on the flow cell
molecules to
• Single DNA strands of a library bind to the adaptor sequence surface
on the flow cell
• Samples are amplified to form clusters
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Illumina Sequencing Workflow

 Created by Commercial Training and Enablement

Illumina Sequencing Workflow

Step 3: Sequencing

Add Scanning Add

Add Scan
Incorporation added Cleavage
Mix
Mix base Mix
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Illumina Sequencing Workflow

Step 3: Sequencing for MiSeq and HiSeq

• Each of the four DNA bases emits an intensity of a unique wavelength

A image
G T A C
C image
Intensi

G image
ty

T image

Wavelength

• After imaging is complete for one section (tile), the flow cell is moved to the next tile and the process is
repeated
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Illumina Sequencing Workflow

Sequencing Paired-End Libraries with Dual Index Reads

Dual Indexed Sequencing Utilizes four Sequencing Reads

1 2 3 4

Paired-End
Turnaround
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Illumina Sequencing Workflow

Step 3: Sequencing

A
G
C
T

Add Scanning Add

Add Scan
Incorporation added Cleavage
Mix
Mix base Mix
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Illumina Sequencing Workflow

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Step 4: Data Analysis

Sequencing Primary Analysis Secondary Analysis Data Visualization

Software Software Software Software

Instrument Control Instrument Control BaseSpace, Local Illumina standalone
Software Software Run Manager (LRM), options, BaseSpace,
DRAGEN or third-party software
Real-Time Analysis Real-Time Analysis
(RTA) (RTA)

Output Output Output Output

Images Intensities Alignments and Charts and Graphs
Variant Detection

Base Calls
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Illumina Sequencing Workflow

Library Prep Cluster
Sequencing Data Analysis
Generation

Genome
Eg: A book with 3
billion letters The book is 1000+ copies Each strip is Now we have
(1,500,000 pages) shredded into of each shred read to reassemble
strips with the book
about 500
letters

This is really the best way to do sequencing

This is really the best way to do sequencing

is really the

the best way to

way to do sequencing