IILM College of Engineering and Technology

Next Generation Sequencing- Revolutionizing the Global Scenario in Genomics Technologies

Completion of Human Genome Project in April 2003, a project initiated by National Human Genome Research Institute, led to a revolutionary change in domains of Genomics and personalized medicine. Deciphering the sequence of entire 3 billion base pairs of human genome was a landmark achievement of International Human Genome Sequencing consortium & Celera Genomics group and they took only 13 years to complete this project. This research Project led to discovery that only 3% of our genome is made of up of coding genes and rest 97% comprises of Junk DNA made up of transposons, tandem repeats, Introns and other non coding regions.

DNA sequencing is the technique of finding the correct sequence of nucleotides A,T,G,C in a given genetic fragment. First generation sequencing started way back in 1970’s when two methods were told by two groups of Scientists. Sanger’s dideoxy Chain Termination method and Maxam Gilbert Sequencing are the techniques, which emerged in 1977 , based on using gel electrophoresis, using fluorescently tagged ddNTPs and terminating the sequencing in a manner that generated sequences vary in just one nucleotide base pair. First complete genomic sequence to be completed was of Bacteriophage phiX174. Continuing with evolution in DNA sequencing technologies, Dr.Pal Nyren at the Royal Institute of Technology in Stockholm published the methodology of Pyrosequencing. Developments in tools of Bioinformatics led to emergence of faster and more inexpensive techniques of DNA sequencing. Whole Genome sequencing using Shotgun Sequencing approach marked a new era in this domain.

Next Generation Sequencing (NGS) is a powerful platform and tool which allows sequencing of thousand to millions of genomes simultaneously. The resolution and extremely high throughput sequencing efficiency provided by NGS has made it a buzz word across the globe. NGS has transformed fields like diagnosing genetic diseases, pharmacogenomics and Clinical diagnostics.

Sanger Sequencing method, developed in 1970s is a golden benchmark methodology which is still used today in routine sequencing applications and validation of NGS data. NGS machines have been introduced in last decade with varying technical details and working principles. Common steps applied during NGS by all these methods involves Sample preparation where DNA is fragmented and ligated with adaptor molecules , followed by Sequencing where all the adaptor ligated DNA fragments are amplified in multiple copies leading to generation of cluster of amplified DNA molecules followed by generation of raw data output which is analyzed further to decipher the correct sequence.

NGS platforms differ in the type of Sequencing techniques adopted during the 2nd step. Mainly four Sequencing methods are applied in NGS- Sequencing by Synthesis, Sequencing by ligation, Pyrosequencing and Ion semiconductor sequencing. ILLumina HiSeq 4000 machines utilizes sequencing by synthesis method, which is the most popular method globally and it is able to generate hundreds of coverage per run. In fact, with this technology, Complete DNA sequencing of 15 individuals can be done simultaneously and will be completed in just 3 and a half day.

NGS has provided us a really powerful tool and we do not lie far away from the day when each individual will have their complete genome card in form and size of an ATM card in their pockets and Doctors will prescribe the medicines based on our specific genome card in every nook and corner of the world.

Written by: Dr. Pallavi Singh, Associate Professor, Department of Biotechnology



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