Next Generation Sequencing


What is Next Generation Sequencing?

Next Generation Sequencing  uses  a set of new high throughput technologies which  allow millions of short DNA sequences from a biological sample to be “read” or sequenced in a rapid manner. The computational power is then used to assemble or align the “reads” to a reference genome, allowing biologists to make comparisons and interpret various biological phenomena very easily . Due to high depth of coverage , accurate  sequencing data  is obtained much faster and cheaper compared to traditional Sanger/Shotgun sequencing

What is Traditional Sanger/Shotgun sequencing?

Sequencing started   in small microbial species in in seventies using Sanger (dideoxy) sequencing method. A variation of this method that involves fragmenting DNA into millions of shorter pieces and sequencing them as “reads” of 50-1000 bases is called Shotgun sequencing . Sequence fragments need to be “assembled”and this is called denovo DNA assembly

Sanger sequencing

Sanger sequencing, also known as chain-termination sequencing, refers to a method of DNA sequencing developed by Frederick Sanger in 1977. This method is based on amplification of the DNA fragment to be sequenced by DNA polymerase and incorporation of modified nucleotides specifically, dideoxynucleotides (ddNTPs).
The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs) that terminate DNA strand elongation . These chain-terminating nucleotides lack a 3′-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently labelled for detection in automated sequencing machines.The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. This is frequently performed using a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C). The DNA bands may then be visualized by auto radiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image.In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths .
Automated DNA-sequencing instruments (DNA sequencers) can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a day. DNA sequencers carry out capillary electrophoresis for size separation, detection and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms. Automation has lead to the sequencing of entire genomes.
Comparison between NGS versus Sanger sequencing

Sanger sequencing is laborious, time-consuming and expensive – yet, it is the only way to assemble full-length genomes and Impossible to process hundred and thousands of samples

NGS is cheap in comparison and very automated .Once genome sequence is available and short reads for a sample generated, alignment is a relatively simple operation, and leads to many more interesting results such as Copy Number Variations, SNPs, Methylation patterns, Gene Expression profiles, etc