Sequencing Technologies

From fragments to reads
Replication
• Replication can be achieved in
vitro if template DNA, Primer,
Polymerase enzyme and
nucleotides are available.
• Nucleotides are added
one at a time
Fragmentation and amplification
• Isolated DNA can be sheared into fragments
either with known ends or unknown ends
• The fragments can be separated into libraries
• Amplified into millions of copies by cloning or
polymerase chain reaction (PCR)
• PCR involves Denaturation, Annealing and
Elongation
Genetic map
Type of
Chromosome map that shows
the relative locations
of genes and other important
features.
Knowledge of it
ensures that the
sequencing
process can be
tailored
Genomic library
A genomic library is
usually stored as a set of
bacteria, each carrying
a different fragment of
human/any species DNA.
Hybridization
Single strand DNA can

pairup with any
complementary strand.
This forms basis of
sequencing techniques
like southern blotting,
Illumina, DNA Array
Sanger Sequencing
• Dideoxy or chain terminataion method
• Fred Sanger, 1977, X174 virus, 5386bp
• Small genome like that of virus succesfully
sequenced
• Based on in vitro DNA synthesis performed in
the presence of chain-terminating nucleotides
(dideoxy nucleoside triphosphates)
• ddNTPs: ddATP, ddGTP, ddCTP, and ddTTP
dideoxy nucleoside triphosphates
Sanger Sequencing Steps
• DNA fragments separated into single strand
• Primer attached to the sequence
• Polymerase solution, four types of dNTPs
(nucleotides) & ddNTP are added.
• Replication process in fragments are stopped as
soon as one of the ddNTP attaches.
• Fragments are separated by gel electrophoresis
and the sequence is determined.
Fragment and ddNTP
• Very accurate, 99.99% base accuracy
• Considered “gold standard” for validating
DNA sequences
• Smaller genome sequenced, cost effective
• Cheap 3$ per cycle
• Most widely used sequencing method for
approximately 40 years
• Read length maximum upto 500 bp
• Slow, becomes expensive for larger and
complex genome
DNA arrays
• Radoje Drmanac, Andrey Mirzabekov, and
Edwin Southern, 1988
• Goal of cheaply generating a genome’s k-mer
composition
• A smaller read length k approx 10bp
Working DNA arrays
• First synthesize all 4k possible DNA k-mers. For
a k mer of length 10bp, the possible k mers to
be synthesized is 4 = 1,048, 576
• Attach them to a DNA array, which is a grid on
which each k-mer is assigned a unique position
• Solution with unknown single strand DNA
fragment with fluorescent label was applied to
the DNA array.
• Unknown fragment hybridizes with the
complementary k mer on array
Reading the array
• Fluoresence was analyzed
with spectroscopy
• The reverse complements
of k-mers corresponding
to these sites belong to
the (unknown) DNA
fragment
• k-mers on the array
reveals the sequence of a
DNA fragment
Array technology application
• Could not be applied to genome sequencing because
Fidelity of DNA for hybridization with the array was too
low and value of k was too small
New Application
• arrays are used to measure gene expression
• Analyze genetic variations.
• DNA arrays is a multi-billion dollar industry that
included Hyseq, founded by Radoje Drmanac (one of
the original inventors of DNA arrays.)
NGS – Next generation sequencing
• Illumina
• Nanopore techniques
Sample preparation
1. Breaking and adaptor attachment
2. Fragment
with adaptor
3. Achment of
bridging group
Cluster Generation
• Clustering is amplification process in flow cell
• Each fragment is first attached onto glass
channels on a flow cell and then amplified into
millions of copy
Sequencing
• Begins by adding one nucleotides at a time
which generates a signal
• The reads are generated for forward as well as
reverse strand
• Illumina generates
paired reads
that is two reads for
each fragment
Data Analysis
• Preliminary data analysis is done
• Data is locally clustered based on indices given to
each cluster
• Contiguous sequence prepared
• Contig aligned to reference genome for
verification and identification.
Nanopore Sequencing
• Unique & scalable technology
• Enables direct, real-time analysis of long DNA or
RNA fragments.
• It works by monitoring changes to an electrical
current as nucleic acids are passed through a
protein nanopore.
• The resulting signal is decoded to provide the
specific DNA or RNA sequence.
• Advantage is fast and cost effective &
disadvantage is error in reads

Sequencing Technologies

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Sequencing Technologies

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From fragments to reads

Single strand DNA can

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