The principle behind Next Generation Sequencing (NGS) is similar to that of sanger sequencing which relies on capillary electrophoresis. The genomic strand is fragmented, and the bases in each fragment are identified by emitted signals when the fragments are ligated against a template strand

NEXT GENERATION SEQUENCING
Iqra Rehman
Iqra Rehman

Next Generation Sequencing

Keywords: DNA sequencing, Next Generation Sequencing, Maxim-Gilbert Sequencing, Sanger Sequencing, 454 Sequencing, Ion Torrent Sequencing, Applied Biosystem Solid Sequencing, Illumine Sequencing, Data Analysis, PCR

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DNA Sequencing;

DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method that is used to determine the order of four bases- adenine, guanine, cytosine and thymine in a stand of DNA and to analyze gene structure and its relation to gene expression as well as protein conformation.

Purpose of DNA Sequencing;

By DNA Sequencing we can compare genes or specific sequences to find out differences and similarities.

Classify organism, make a disease diagnosis

Through the DNA sequencing would be able to know where exactly into genome or a gene is the mutation.

Basic Methods for DNA Sequencing;

Maxam-Gilbert sequencing

Sanger sequencing

Maxam-Gilbert Sequencing;

This method is based on the nucleobase-specific partial chemical modification of DNA and subsequent cleavage of DNA backbone at the sites adjacent to the modified nucleotides.

Sanger Sequencing

Sanger sequencing also known as chain termination sequencing or dideoxy sequencing. This process is based on the detection of labeled chain-terminating nucleotides that are incorporated by a DNA polymerase during the replication of a template.

Developed by Frederick Sanger and colleagues in 1977. This method determining the location of a specific place on a DNA fragment, based on where synthesis of a new DNA chain stops.

Principle;       

Utilizes 2’,3’-dideoxynucleotide triphosphate (ddNTPs) and different from dNTPs at the 3’carbon have a hydrogen atom attached to the 3’carbon rather than an OH group.

Next Generation Sequencing:

It is non-Sanger-based high throughput DNA sequencing technologies. Millions or billions of DNA strand can be sequenced in parallel.

Principle of Next Generation Sequencing:

“The principle behind Next Generation Sequencing (NGS) is similar to that of sanger sequencing which relies on capillary electrophoresis. The genomic strand is fragmented, and the bases in each fragment are identified by emitted signals when the fragments are ligated against a template strand.”

It is non-Sanger-based high throughput DNA sequencing technologies. Millions or billions of DNA strand can be sequenced in parallel

Basic Steps of Next Generation Sequencing:

Library preparation:

Fragmentation of genome: First DNA is fragmented either enzymatically or by physical shearing and creates a smaller DNA fragment.

Ligation of Adaptors:  Adaptors are short piece of synthetic DNA. They are ligated to these fragments with the help of DNA ligase.

Amplification:

The library is amplified by using clonal methods and PCR.

Attachment of Targeted DNA to the Beads:  Beads are insoluble molecules which are not interacting with other chemicals or enzymes. Beads are constructed in such way that each bead is surrounded by a known single stranded nucleotides sequence. Adaptors are attached to these sequences which present on bead. Beads carry those sequences which is complementary to adaptor.

Loading beads into wells: After attachment of adaptor on beads we start adding nucleotides to produce complementary sequence if once a complementary structure produce it will go and again bind to other beads which is complementary in nature and this process go on and it will produce multiple copies of DNA. This whole process done in a well. We run the amplification process by just adding the nucleotides time to time and DNA polymerase

Emulsion PCR: Emulsion PCR is a method of clonal amplification. Emulsion oil, beads, PCR mix and the library DNA are mixed to form an emulsion which leads to formation of micro wells.

Bridge PCR:  In this type surface of the flow cells is densely coated with primers that are complementary to the primers attached to the DNA fragments.

Sequencing:

In Next Generation Sequencing DNA is sequenced by four main types of sequencing.

454 sequencing

Ion torrent sequencing

Sequencing by ligation (SOLID)

Reversible terminator sequencing (Illumina)

454 Sequencing:

It is fast and reliable technique and it depend on fluorescence data to sequence the DNA. First larger genome is fragmented into small fragments and then attaches two types of adaptors. In 454 sequencing the adaptor A and adaptor B are used after ligation of adaptors on fragments these adaptors attach to the bead’s adaptor A will attach on beads. Its sequence is complementary to the sequence of beads.

We will load these beads into micro wells once it put into wells the wells are inserted into actual sequencing machine. Till now all process going on pre- sequencing machine after it we add primer. Primer bind with adaptor B we polymerase this primer by adding nucleotide at 3 end. Each type of nucleotide generates specific fluorescence because we tagged each nucleotide with specific color (like red, green, blue etc.)

We measured the degree of florescence by the way we checked the specific nucleotide present or not in the sequence of targeted DNA. We plot a graph by using data and show into peaks. All data put in CPU through software program process the data of each fragment. We will find the overlapping region of these fragments and then we get the actual whole genome sequence.

Basic Steps of Next Generation Sequencing:
Library preparation:
Fragmentation of genome: First DNA is fragmented either enzymatically or by physical shearing and creates a smaller DNA fragment.
Ligation of Adaptors:  Adaptors are short piece of synthetic DNA. They are ligated to these fragments with the help of DNA ligase.
Amplification:
      The library is amplified by using clonal methods and PCR.
Attachment of Targeted DNA to the Beads:  Beads are insoluble molecules which are not interacting with other chemicals or enzymes. Beads are constructed in such way that each bead is surrounded by a known single stranded nucleotides sequence. Adaptors are attached to these sequences which present on bead. Beads carry those sequences which is complementary to adaptor.
Loading beads into wells: After attachment of adaptor on beads we start adding nucleotides to produce complementary sequence if once a complementary structure produce it will go and again bind to other beads which is complementary in nature and this process go on and it will produce multiple copies of DNA. This whole process done in a well. We run the amplification process by just adding the nucleotides time to time and DNA polymerase 
Emulsion PCR: Emulsion PCR is a method of clonal amplification. Emulsion oil, beads, PCR mix and the library DNA are mixed to form an emulsion which leads to formation of micro wells. 
Bridge PCR:  In this type surface of the flow cells is densely coated with primers that are complementary to the primers attached to the DNA fragments.

Sequencing:
In Next Generation Sequencing DNA is sequenced by four main types of sequencing.
454 sequencing
Ion torrent sequencing
Sequencing by ligation (SOLID)
Reversible terminator sequencing (Illumina)

454 Sequencing:
It is fast and reliable technique and it depend on fluorescence data to sequence the DNA. First larger genome is fragmented into small fragments and then attaches two types of adaptors. In 454 sequencing the adaptor A and adaptor B are used after ligation of adaptors on fragments these adaptors attach to the bead’s adaptor A will attach on beads. Its sequence is complementary to the sequence of beads.
We will load these beads into micro wells once it put into wells the wells are inserted into actual sequencing machine. Till now all process going on pre- sequencing machine after it we add primer. Primer bind with adaptor B we polymerase this primer by adding nucleotide at 3 end. Each type of nucleotide generates specific fluorescence because we tagged each nucleotide with specific color (like red, green, blue etc.)
We measured the degree of florescence by the way we checked the specific nucleotide present or not in the sequence of targeted DNA. We plot a graph by using data and show into peaks. All data put in CPU through software program process the data of each fragment. We will find the overlapping region of these fragments and then we get the actual whole genome sequence.

Ion Torrent Sequencing:

It is another type of next generation sequencing in which no light and fluorescence is used and it is cheaper and faster than many of other method. It is based on proton which is release when phosphodiester bond is formed. Basic step is same in this type of NGS.

First is fragmentation of genome then ligation of adaptors after its next step is attachment of adaptors on beads and next step is loading of beads into wells. Microchip has millions or billions of tiny wells each well is load with one bead. This microchip is a heart of Ion Torrent sequencing. This chip has a secondary layer which is known as ion sensitive layer after loading the well we start adding nucleotide in all wells at a time and same nucleotide is added in all wells.

Where complementary base is present this new base is pair up and release a hydrogen ion. When proton release then pH is change and this change pH is sense by an ion sensitive layer. Each tiny well has tiny pH meter so, millions of pH meter combine and functioning together. The advantage of this type is that the chip used in it have enormous capability whole genome runs at once now we have chip of 100GB and it take 2-3 hours for accurate sequencing of genome.

Applied Biosystems SOLID Sequencing

(Sequencing by Oligonucleotide Ligation and Detection). It is next generation DNA sequencing technology developed by Life technologies and has been commercially available since 2006. This technology generates hundreds of millions to billions of small sequences reads at one time.

Mechanism of SOLID sequencing:

DNA fragmented library is used which is flanked by legated adaptors. Fragments are attached to small paramagnetic beads and emulsion PCR is performed to amplify the fragments. Other method rather than polymerase is also used that is utilizing DNA ligase. Each cycle of sequencing involves the ligation of degenerative population of fluorescently labeled universal octamer primers. Octamer is fluorescently labeled.

After ligation, images are acquired in four channels, followed by cleavage of octamer b/w position 5 & 6. After several round of octamer ligation, which enable sequencing of every 5th base (e.g.; 5, 10, 15 &20). Different primers used to examine next and previous position. This approach is involving examination of bases twice in a cycle, which decreases the error rates. SOLID read lengths are 25-35bp and each sequencing run yield is 2-4Gb of DNA sequence data.

It is next generation DNA sequencing technology developed by Life technologies

Illumine sequencing

It is technique used to determine the series of base pairs in DNA. The reversible terminated chemistry concept was invented by Bruno canard and simom sarfati at the Pasteur institute in Paris. it is frequently used for NGS. It is used for RNA sequencing. 

Steps to ILLUMINA sequencing

Library construction: fragments, attach adapter DNA.

Cluster generation: at to flow cell, bridge amplification.

Sequencing: single base at a time, imaging.

Data analysis: image transform into basecall and reads.

Advantages of Next Generation Sequencing:

High degree of Parallelism then Capillary Sequencing

Low Reagent Cost

Reduced Sample Size

Less Time Consuming

Can also sequence transcriptomes

  Disadvantages of Next Generation Sequencing:

  Next Generation Sequencing is still too expensive.

  Inaccurate sequencing of homopolymer regions.

  Data analysis can be time-consuming.

  Require special knowledge of bioinformatics

 Applications of Next Generation Sequencing:

 Whole genome re-sequencing

 Metagenomic

 Epigenetics

 Gene regulation

 RNA sequencing

Applications of Next Generation Sequencing
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