Next Generation Sequencing

Sequencing is a method to identify the exact order of the nucleotides present in a DNA sample.

Sanger sequencing:

• Chain termination method of sequencing DNA.

DNA polymerase
All four nucleotides (C,T,G,A)
One dideoxynucleotide (ddNTP)

Random termination of the chain occurs at specific bases where ddNTP (tagged to a fluorochrome) is incorporated instead of dNTP.
Separated by gel electrophoresis.


Next Generation Sequencing: Also known as high-throughput sequencing

• Millions of fragments of DNA from a sample are sequenced at one time using parallel sequencing technology.
• Facilitates high-throughput sequencing, and allows entire genome to be sequenced in less than one day. 
• Massive parallel sequencing by synthesis and ligation

NGS:

• During sequential synthesis of DNA, dNTPs which are fluorescent tagged) are incorporated. This is catalysed by DNA polymerase.
• They are identified by fluorophore excitation at the point of incorporation.
• Important difference between NGS and other systems of DNA sequencing: Millions of fragments of DNA are sequenced in a parallel fashion. 

Applications:

• To identify genes and regulatory elements involved in disease processes.
• Whole-genome sequencing
• Exome sequencing
• Targetted sequencing

Platforms:

• Illumina Sequencing - detects the emission of fluorescent signal during addition of a base/ nucleotide.
  
• Ion Torrent/ Proton sequencing - detects the release of protons (H+) during incorporation of every nucleotide in the DNA.
  
• Roche Sequencing - Based on pyro-sequencing technology - detects the release of pyrophosphate.

Disadvantages:

• Huge cost
• Cumbersome to analyse the data generated
• Time consuming
• Lack of knowledge on bio-informatics  to analyse the data.