The history of DNA sequencing. Let’s take a journey from the first isolation of DNA to next generation sequencing (NGS). Read more.

1. EUROFINS GENOMICS
The DNA Universe

2. 2
DNA sequencing is the determination of the nucleotide or base sequence
(adenine, guanine, cytosine, thymine) in a DNA molecule.
What is DNA sequencing to begin with?
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DNA sequencing is the key analytical method for:
• Cloning
• Investigation of genetic diseases
• Genotyping

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1965 Robert Holley sequences the first tRNA
1972 Walter Fiers sequences the DNA of a complete gene (coat
protein of the bacteriophage MS2) by utilising RNAses to digest the
virus RNA and isolate oligonucleotides, and then separating them
via electrophoresis.
1977 Fredrick Sanger developes the first DNA sequencing
method that utilises radiolabelled partially digested fragments:
Chain termination method.
Sanger sequencing dominates the field of genetics for
three decades.
“Scientific research is one of the most exciting and rewarding of occupations. It is
like a voyage of discovery into unknown lands, seeking not for new territory but for
new knowledge. It should appeal to those with a good sense of adventure”
- Fredrick Sanger
It really started with Sanger sequencing
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tRNA
MS2 Capsid

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Sanger sequencing principle
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The sequencing reaction uses
deoxynucleoside triphosphates
(dNTPs) and modified
dideoxynucleoside triphosphates
(ddNTPs) for strand elongation.
ddNTPs are fluorescent label and
with a chemical group that causes
DNA polymerase to stop DNA
extension when ddNTP is
incorporated.
Resulting DNA fragments are
analysed by capillary
electrophoresis.
The fragments flow through
capillary at different speeds
according to their size.
The nucleotides are determined by
fluorescence signal.

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1984 Fritz Pohl establishes the first
sequencing technology platform that does not
rely on radioactive labelling: the GATC1500.
Progress in DNA sequencing
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GATC1500
ABI 370
1987 Leroy Hood and Michael
Hunkapiller succeed in the automation of
the Sanger sequencing process:
• DNA fragments are labelled with
fluorescent dyes
• Data acquisition and analysis is done
with a computer
• Device is called ABI 370.
1995 First bacterial genome is
sequenced: Haemophilus influenza.

6. 6
1996 Mostafa Ronaghi, Mathias Uhlen and Pȧl Nyŕen introduce
pyrosequencing.
Pyrosequencing is based on measurement of luminescence generated as a result
of pyrophosphate synthesis during sequencing (sequencing-by-synthesis
technology).
This is considered as the emergence of the second generation of DNA sequencing.
Second generation of DNA sequencing
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S. cerevisiae
1996 Genome of Saccharomyces cerevisiae
(yeast) is sequenced.
1996 ABI introduces first commercial
sequencing using capillary electrophoresis.

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1998 Development of
sequencing-by-synthesis method
that uses fluorescent dyes.
1998 Genome of Caenorhabditis
elegans is sequenced.
1999 First human chromosome 22 is
sequenced.
2002 Complete mouse genome is
sequenced.
Second generation of DNA sequencing
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C. elegans Human
chromosome 22
Sequencing-by-synthesis principle

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Sample/library preparation:
A library is prepared by fragmenting DNA
sample and ligating DNA with adapters:
• Adapter molecules act in hybridisation of
library fragments to matrix.
• Adapter molecules provide priming site.
Amplification and sequencing:
• Library is converted into single stranded
molecules.
• Amplification creates clusters of DNA
molecules.
• Each cluster acts as an individual reaction
where sequencing, called run, is performed.
Data output and analysis:
• Each NGS run provides a large amount of raw
data.
• This data can be analysed by using a variety
of available software.
Sequencing-by-synthesis principle
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Differences among available NGS technologies lie in the
details of amplification and sequencing reaction. PacBio
SMRT does not involve any amplification step.
Sample/library preparation

9. 9
2005 Jonathan Rothberg and colleagues
implement the pyrosequencing technology in an
automated system: The 454 system, first NGS
platform to come to market.
2007 SOLiD system introduces “sequencing-by-ligation”.
It uses DNA ligase to identify the nucleotides in DNA
sequence.
2008 First complete DNA sequence of a cancer is
decoded.
2011 Life Technologies introduces Ion Torrent that uses
“sequencing-by-synthesis” technology and detects
hydrogen ions when new DNA is synthesised.
Second generation of DNA sequencing
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Roche 454 Sequencing
System
Ion Personal Genome
Machine

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2011 Pacific Biosciences (PacBio) introduces
zero-mode waveguide (ZMW) that utilises
“nanoholes” containing a single DNA polymerase.
Incorporation of single nucleotide (labelled with
fluorescent dyes) can be observed directly.
‘‘We can multiplex that process and observe this phenomenon as it
happens over thousands or tens of thousands of these holes
simultaneously. The DNA polymerase becomes the sequencing engine.
That is a true revolution.” -Eric Schadt, PacBio CSO
2012 Oxford Nanopore Technologies launches portable
system for RNA and DNA sequencing: GridION. Other
handheld systems such as MinION follow.
The system uses changes in electrical conductivity that
occur when DNA strands pass through biological
nanopores to identify nucleotide sequence.
Third generation of DNA sequencing
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PacBio RSII sequencer
Oxford Nanopore
Technologies MinION

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Eurofins Genomics whole product portfolio is structured to
address 3 types of customer needs
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BUILT
FOR YOU
Pre-designed and validated
packages for standard
applications
BUILD
YOUR OWN
Flexible standardized
modules for easy design
of custom solutions
CUSTOMISED
SOLUTIONS
Tailored solutions for
complex projects, designed
together with our experts
DNA & RNA
Oligonucleotides
Application oligos and
qPCR probes
Flexible modules to design
custom DNA and RNA oligos
e.g., custom qPCR assay
with primers and probe in
plates
Custom DNA
Sequencing
Simple premixed solutions in
tubes and plates
Flexible options for
sequencing in tubes and
plates
e.g., SNP analysis, primer
walking, GLP sequencing
Next Generation
Sequencing
INVIEW packages for
frequent applications
Flexible modules for DNA,
RNA and amplicon
sequencing
e.g., GLP sequencing,
custom amplicon panel
Gene Synthesis /
Molecular Biology
Synthetic genes and
site-directed mutagenesis
Modules of gene synthesis
for individual ordering (e.g.,
gene fragments)
e.g., super express gene
synthesis, plasmid
linearization
Genotyping &
Gene Expression
CLA for standard cell lines
and mycoplasma check
CLA for custom cell lines and
FLA
e.g., microarray analysis,
authenticity testing

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