Department of Biotechnology & Genetic Engineering

Jahangirnagar University
Savar, Dhaka

Assignment On Next Generation Sequencing

Submitted To :

Dr. Farah Shamma

Associate Professor
Department of Biotechnology &
Genetic Engineering

• Submitted By :

Kaniz Fatema-1576
Md. Tanjim Hasan-1577
Rifat Ahmed Shakil-1579
Abu Rahad-1585
Nadir Hossain Sunny-2242
Group No: 02

Submission Date: 10.11.23

1. Pyrosequencing steps, principle, why massively
parallel sequencing is possible using
pyrosequencing?

Ans: Pyrosequencing is a method for DNA sequencing. Pyrosequencing in used for high-
throughput DNA sequencing. It is a method for sequencing DNA thus it determines the order
of nucleotides a segment of DNA. Pyrosequencing is based of sequencing by synthesis
principle. In this method, sequencing is performed by detecting the nucleotides incorporated
by a DNA polymerase. This relies on light-based detection technique where a pyrophosphate
is released upon addition of a nucleotide.

Principle:

The basis of the technique is the detection of pyrophosphate that is released during DNA
synthesis. As part of the structure of a deoxy nucleoside triphosphate, the phosphate attached
to the 5′-carbon of the deoxyribose sugar moiety is the α-phosphate, the next phosphate is the
β-phosphate, and the third is the γ-phosphate. During replication, the α-phosphate of each
incoming complementary nucleotide is joined enzymatically by a phosphodiester linkage to the
3′-OH group of the last nucleotide of the growing strand, and the β - and γ -phosphates are
cleaved off as a unit that is called pyrophosphate.

The strategy for pyrosequencing requires a series of enzymatic reactions. Briefly, the
pyrophosphate generated by the incorporation of a nucleotide by DNA polymerase (in DNA
polymerization reaction) is combined with adenosine-5′-phosphosulfate (APS) by the enzyme
ATP sulfurylase to form ATP. In turn, ATP drives the conversion of luciferin to oxyluciferin
by the enzyme luciferase, which generates visible light in amounts that are proportional to the
amounts of ATPs. That means, the amount of light generated after the addition of a particular
nucleotide tends to be proportional to the number of nucleotides that are incorporated in the
growing strand. The light in the luciferase-catalyzed reaction with a maximum of 560
nanometer wavelength. It is detected by a photon detection device such as a charge coupled
device (CCD) camera or photomultiplier.
Figure 1: Biochemical reactions of pyrosequencing.

• Steps involved in Pyrosequencing

STEP 1 - INCUBATION OF TEMPLATE STRAND WITH THE PRIMER

The DNA fragment of unknown sequence is taken. This DNA sequence is engineered at one
end that is complementary to a known primer. This DNA fragment serves as DNA template
strand and it is incubated with the primer. The primer binds to its complementary sequence on
the DNA template strand.
Figure 2: Pyrogram.

STEP 2 - ADDITION OF ENZYMES AND SUBSTRATE

In this step DNA polymerase is added, along with the other enzymes (ATP sulfurylase,
Luciferase) and substrates (Adenosine phosphor sulphate, luciferin) required for the detection
of pyrophosphate.

STEP 3 - ADDITION OF ONE TYPE OF NUCLEOTIDE

After the second step one of the four types of nucleotides is added. Only one type of nucleotide
is added at a time. That means if we are adding dCTP, then the solution of nucleotide we are
adding contains only dCTP molecules.
If the added nucleotide is incorporated in the new strand, pyrophosphate will be released and
emission of light will take place. This light is detected by a detector and later used to interpret
the unknown sequence. But if the added nucleotide is not incorporated, then there will be no
pyrophosphate and therefore no light emission. This happens when the incoming nucleotide is
not complementary to the nucleotide of the template strand.

STEP 4: ENZYMES APYRASE IS ADDED FOR REMOVES UNUSED

NUCLEOTIDES
Now in both the cases whether the nucleotide is incorporated or not. Extra or unused nucleotide
will be present. The extra nucleotides are now removed from the reaction. This is done by
adding enzyme apyrase the nucleotide degrading enzyme.

STEP 5 - REACTION STARTS AGAIN WITH ANOTHER NUCLEOTIDE

After the degradation of unused and extra nucleotides is completed. The pyrosequencing
reaction starts again with the addition of next nucleotide.
This process is repeated adding each nucleotide one after the other until the synthesis is
complete. So, every time a complementary nucleotide is added emission of light will take place.

Pyrosequencing for High-throughput Sequencing:

Pyrosequencing is only able to generate up to 150 bp in a single experiment. This might appear
to be less useful than the chain termination method, especially if the objective is to sequence a
genome. The advantage with pyrosequencing is that it can be automated in a massively parallel
manner that enables hundreds of thousands of sequences to be obtained at once, perhaps as
much as 1,000 Mb in a single run. Sequence is therefore produced much more quickly than is
possible by the chain termination method, which explains why pyrosequencing is gradually
taking over as the method of choice for genome projects.
2. Techniques-Single end, Paired-end, Mate-pair sequencing

• Single-end Sequencing
Single-end sequencing involves a series of steps to prepare the DNA sample for sequencing.
Initially, the DNA sample is fragmented into fragments ranging from 200 to 500 base pairs
(bp) in length. A primer sequence is then attached to one end of each DNA fragment. After that
the addition of a splice is done to facilitate subsequent processing. These prepared fragments
are immobilized on a flow cell, generating a DNA cluster ready for sequencing. The sequencing
process itself involves reading the DNA sequence from one end of the fragment, thus yielding
a single-end read.

Advantages:

1. Simple and efficient

2. Requires fewer steps for library preparation.

Limitations:

1.As the sequencing process progresses, the quality of the reads tends to degrade.

2. Increased inaccuracies in the later portions of the sequence.

 • Paired-end Sequencing

Paired-end sequencing involves the construction of a specialized DNA library designed for
enhanced sequencing accuracy. This library is created by incorporating sequencing primer
binding sites at both ends of the DNA junction. The sequencing process then proceeds in two
rounds. In the initial round, the template strand from the first sequencing round is removed,
and the complementary strand is regenerated and amplified at its original position using a
specialized module called the Paired-End Module. This amplification step ensures an adequate
supply of template DNA for the second round of sequencing. The second round involves
synthesizing the complementary strands, yielding paired-end reads.

Figure 3: Pair-end sequencing.

• Mate Pair Sequencing

Following DNA fragmentation, the DNA fragments are end-repaired with labeled dNTPs. The
DNA fragments are circularized, and non-circularized DNA is removed by digestion. Circular
DNA is fragmented, and the labeled fragments (corresponding to the ends of the original DNA
ligated together) are affinity-purified. Purified fragments are end-repaired and ligated to
Illumina paired-end sequencing adapters.

Additional sequences complementary to the flow cell oligonucleotides are added to the adapter
sequence with tailed PCR primers. The final prepared libraries consist of short fragments made
up of two DNA segments that were originally separated by several kilobases. These libraries
are ready for paired-end cluster generation, followed by sequencing utilizing an Illumina next-
generation sequencing (NGS) system.

Figure 4: Mating pair sequencing.

3. Cluster Generation

In next-generation sequencing (NGS), cluster generation is a crucial step in the sequencing

process. This process is especially related to the Illumina sequencing platform, which is one of
the most extensively used NGS technologies. Cluster generation is critical because it ensures
that each cluster on the sequencing surface corresponds to a single DNA fragment. This enables
the accurate and parallel sequencing of millions of DNA fragments in a massively parallel
fashion, a hallmark of next-generation sequencing technologies like Illumina.

➢ Cluster Generation Process:

The prepared library is loaded into a flow cell and placed in the sequencer. A flow cell is a
glass slide with 1, 2, or 8 physically separated lanes coated with a lawn of surface-bound,
adapter-complimentary oligos. Nowadays, the most often used flow cell is a patterned flow
cell manufactured utilizing semiconductor manufacturing techniques. It has a glass substrate
with patterned nano wells holding DNA probes that capture the prepared DNA strands for
amplification during cluster generation.

Figure 5: Illumina Patterened flow cell.

Cluster generation begins once the samples are attached to the flow cell and bridge
amplification occurs. During this procedure, the DNA fragments from the library hybridized
with one of the oligonucleotides on the flow cell surface. The oligo linked to the flow cell is
subsequently elongated by DNA polymerase to create a complementary strand. The initial
molecule is washed away, and the strand bends over like a bridge and joins to the next oligo in
the flow cell. This second oligo is complementary to another adapter sequence, and polymerase
creates a complementary strand, making a double-stranded bridge. This bridge is denatured,
resulting in two single-stranded copies of the molecule linked to the flow cell. The procedure
is continued indefinitely and simultaneously for millions of clusters, resulting in clonal
amplification of all.

Following bridge amplification, the reverse strands are cleaved and washed away, leaving only
the forward strands. The 3′ ends are plugged to prevent undesired priming. When cluster
formation is complete, the templates are ready for sequencing.

Figure 6: Bridge Amplification Process.

4. Clonal Amplification
Clonal amplification is a crucial step in the next-generation sequencing (NGS) process,
particularly in sequencing platforms like Illumina. This step involves the generation of
numerous identical copies, or clusters, of DNA fragments from a single DNA molecule. The
purpose of clonal amplification is to create enough material for efficient and accurate
sequencing.

Clonal amplification is performed on DNA libraries prior to sequencing reactions. The

libraries' DNA fragments are amplified in this method such that fluorescent signals of single-
base incorporation in the subsequent sequencing reaction are robust enough to be recognized
by the sequencers.

The Illumina platform employs solid-phase amplification, which involves each fragment in the
library annealing to the primers on the sequencing chip (known as the flow cell) via adapters.
Each fragment generates a cluster of identical molecules termed clonal clusters (Figure 3B)
through a sequence of amplification events known as bridge amplification [4]. Each cluster
represents one primary library molecule. It should be noted that clonal amplification on a
patterned flow cell with predetermined arrays uses a separate technique known as exclusion
amplification (ExAmp) chemistry. ExAmp technique utilizes the immediate amplification of a
DNA fragment after attaching to the primer on the patterned flow cell, preventing additional
DNA fragments from forming a polyclonal cluster.

This technique of clonal amplification should not be confused with library amplification, which
is done before loading a library onto a flow cell.

❖ Clonal amplification steps.

(A) Bridge amplification.
(1) The complementary strand of a DNA fragment in the library is synthesized from the flow
cell’s priming oligo. (2) After removal of the original strand, the complementary strand folds
over and anneals with the other type of flow cell oligo. A double-stranded bridge is formed
after synthesis of its complementary strand. (3) The double-stranded bridge is denatured,
forming two single strands attached to the flow cell. (4) The process of bridge amplification
repeats, and (5) more clones of double-stranded bridges are formed.
(B) Cluster generation.
The double-stranded clonal bridges are denatured (only one strand is shown here for
simplicity), the reverse strands are removed, and the forward strands remain as clusters for
sequencing.

Figure 7: Clonal amplification steps.

5. Library Preparation
Library preparation is a crucial step in the next-generation sequencing (NGS) workflow. It
involves the conversion of DNA or RNA samples into a library of fragments suitable for
sequencing. The goal of library preparation is to create a representative collection of nucleic
acid fragments that can be efficiently sequenced and accurately reflect the genetic or
transcriptomic content of the original sample.

❖ Here are the key steps involved in library preparation:

1. DNA or RNA Fragmentation: In the case of DNA sequencing, the genomic DNA is
typically fragmented into smaller, more manageable pieces. This fragmentation can be
achieved through mechanical methods (e.g., sonication), enzymatic methods (e.g.,
restriction enzyme digestion), or a combination of both. For RNA sequencing (RNA-Seq),
the RNA is typically converted into complementary DNA (cDNA) through reverse
transcription before fragmentation.
2. End Repair and A-Tailing: The fragmented DNA often has uneven ends or damaged
termini. End repair enzymes are used to create blunt ends, and A-tailing involves adding
an adenine (A) overhang to the 3' end of each fragment. These processes ensure
compatibility with subsequent adapter ligation.
3. Adapter Ligation: Adapters are short, double-stranded DNA molecules with sequences
that are compatible with the sequencing platform. These adapters are ligated to the ends of
the DNA fragments. They serve as priming sites for the amplification and sequencing steps.
Unique barcodes may be incorporated into the adapters to allow for sample multiplexing.
4. Size Selection: After adapter ligation, the library is often subjected to size selection to
remove undesired fragments, such as adapter dimers or excessively long fragments. Gel
electrophoresis & other size-selection techniques may be employed.
5. Amplification (Optional): The library may undergo amplification to increase the amount
of DNA available for sequencing. Polymerase chain reaction (PCR) is commonly used for
this purpose. However, care must be taken to avoid over-amplification, which could
introduce biases and artifacts.

Once the library preparation is complete, the prepared samples are ready for loading onto the
sequencing platform. The libraries are then clonally amplified (cluster generation), and the
sequencing process begins.
Figure 8: Overview of some commercially available library preparation protocols. The colors
(red, blue, green and yellow) indicate which protocol steps require similar automation
demands. Each block includes several pipetting and mixing steps.
6. Adapter Ligation
Adapter ligation is a critical step in the library preparation process for next-generation
sequencing (NGS). Adapters are short, double-stranded DNA molecules with sequences that
are compatible with the sequencing platform being used. The main purpose of adapter ligation
is to attach these adapters to the ends of the fragmented DNA or cDNA molecules, preparing
them for subsequent steps in the sequencing workflow.

Figure 9: he basic structure of an NGS adapter prior to amplification, showing key

functional motifs.

❖ Here's an overview of the adapter ligation process:

• End Repair:
Before adapter ligation, the fragmented DNA or cDNA molecules often undergo end repair.
This step involves the enzymatic repair of the ends of the DNA fragments to create blunt ends.
Blunt ends are essential for the proper ligation of adapters.

• A-Tailing:

After end repair, an A-tailing step is often performed. This involves adding a single adenine
(A) nucleotide to the 3' end of each DNA fragment. The A-tailing process is crucial for the
subsequent ligation, as many adapters have a complementary overhang at their 3' end.

• Adapter Design:

Adapters are designed to have sequences that can hybridize with the repaired and A-tailed ends
of the DNA fragments. The adapters typically contain sequences that are compatible with the
sequencing platform's chemistry and allow for the subsequent steps in the sequencing process,
such as cluster generation and sequencing-by-synthesis.

• Adapter Ligation:

The adapters are ligated to the DNA fragments using DNA ligase enzymes. DNA ligase
catalyzes the formation of phosphodiester bonds between the 3' end of the DNA fragments and
the 5' end of the adapters. This ligation step effectively links the adapters to the DNA
fragments, creating a construct ready for sequencing.

• Size Selection (Optional):

Depending on the sequencing application, the library may undergo size selection to remove
undesired fragments. This is especially important for preventing the sequencing of adapter
dimers or overly long fragments. Size selection can be achieved through gel electrophoresis,
magnetic bead-based methods, or other techniques.

Figure 10 : Adapter ligation Process.

7. Base Call
"Base call" refers to the assignment of a specific nucleotide base (adenine, thymine, cytosine,
or guanine, often represented by the letters A, T, C, or G) to a position in a DNA or RNA
sequence during the process of DNA sequencing. In other words, it's the determination of the
identity of a nucleotide at a particular location in a sequence.

During DNA sequencing, the sequencing instrument records signals corresponding to the
incorporation of nucleotides into a growing DNA strand. These signals are then translated into
a sequence of nucleotide bases through a process known as base calling. The base calling
algorithm interprets the raw data, usually in the form of fluorescence signals or other types of
detection signals, and assigns a specific base to each position along the DNA strand.

Accurate base calling is crucial for obtaining reliable sequencing results. Errors in base calling
can lead to inaccuracies in the final sequence data. Different DNA sequencing technologies
and platforms may employ variations in base calling methods, but the general principle is to
convert raw signal data into a readable and accurate DNA or RNA sequence. This sequence
data is then used for various genomics applications, such as genome assembly, variant calling,
and functional genomics studies.

Figure 11: Base Calling Process.

8. Sequencing by Synthesis
Sequencing by synthesis is a technique used in next-generation sequencing (NGS) technologies
to determine the sequence of DNA or RNA molecules. This method relies on the stepwise
addition of nucleotides to a growing DNA strand, with the incorporation of each nucleotide
being detected and recorded.

Every cluster on a flow cell has the same sequence. Sequencing requires the use of a sequencing
primer, DNA polymerase, and fluorescently labelled DNA. However, depending on the
chemistry utilized in their respective machine (4-channel chemistry, 2-channel chemistry, and
1-channel chemistry), either all or simply a few nucleotides are labelled with the fluorophore.

In 4-channel chemistry, all the nucleotides are labeled with four fluorescent dyes. Two-
channel chemistry uses two different fluorescent dyes, while one-channel chemistry uses only
one dye.

In the case of 4-channel chemistry, all of the foregoing components are passed through the
flow cell, where the sequencing primer anneals to its corresponding position on the adaptor and
DNA polymerase adds the fluorophore-labeled complimentary nucleotides. This fluorophore
functions as a blocking group, and DNA polymerase cannot add another nucleotide until it is
removed, allowing the detector to record the fluorescence of each base added. Following the
addition of each nucleotide, the fluorophore in the cluster is activated by the light source, and
the distinctive fluorescence signal emitted is measured. This process is called sequencing-by-
synthesis.

Figure 12: Four-, Two- and One- Channel Chemistry: Four Channel chemistry uses nucleotides
labelled with four different dyes, two channel chemistry uses two different fluorescent dyes
and one channel Chemistry uses only one dye
Figure 13: Sequencing by Synthesis.