DNA sequencing determines the order of nucleotides in a DNA segment. The first generation methods developed in 1977 were Maxam-Gilbert and Sanger sequencing, with Sanger becoming the most common. Next generation sequencing now includes Illumina, which is the most widely used, as well as PacBio and Nanopore third generation real-time single molecule techniques.

2. DNA SEQUENCING
• DNA sequencing is used to determine the order of nucleotides in a
DNA segment, genome, or microbiome.
• The steps needed to obtain the order of nucleotides depend on the
sequencing method chosen, with common techniques including
Sanger, Illumina, PacBio and Nanopore sequencing.
History of DNA sequencing:
• The history of DNA sequencing can be divided into several
generations, each characterized by its own techniques and
technological advancements.

3. First generation sequencing:
• The first major breakthrough in DNA sequencing was achieved in 1977
when two methods – Maxam-Gilbert and Sanger sequencing – were
developed.
• Sanger sequencing proved to be more accurate, robust and easier to
use, so quickly became the most common technology used to sequence
DNA, and remained so for many years. The method is also known as
chain termination sequencing, and relies on the incorporation of
dideoxynucleotides into the DNA strand during replication.

4. Next generation sequencing:
• Sanger sequencing is still used around the world today, but has been
largely superseded by next generation or high throughput sequencing
techniques, which can be divided further into second and third
generation methods.
Second generation sequencing
• The second generation of DNA sequencing began with the
introduction of pyrosequencing by a group from the Royal Institute of
Technology in Stockholm, Sweden, in the late 1990s.

5. • Pyrosequencing is based on the detection of pyrophosphate release
during DNA synthesis. It was the first NGS technology to be
commercialized with the introduction of a sequencer by 454 Life
Sciences in 2005. Today the technology has been largely replaced by
more advanced NGS methods that offer a higher throughput.
• One of these techniques is illumina sequencing. It is based on the use
of reversible terminator-bound dNTPs that are incorporated into the
DNA during synthesis, and has undergone several improvements since
the launch of the first sequencing machine in 2007. Nowadays,
illumina sequencing is the most widely used NGS technology.

6. Third generation sequencing
• As we'll see below, Illumina sequencing requires a DNA amplification
step, which has several disadvantages. Different research groups have
therefore explored real-time, single-molecule sequencing, leading to the
introduction of the two third generation sequencing techniques from
Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT).
• PacBio sequencing monitors the incorporation of nucleotides by a
polymerase enzyme in real-time, whereas Nanopore sequencing uses
nanopores embedded in lipid membranes. When single-stranded DNA
(ssDNA) molecules pass through these pores, electrical signals that vary
depending on the base sequence of the molecules can be detected.

8. DNA SEQUENCING METHODS :