High Throughput Sequencing (NGS) / Next Generation Sequencing

BioCodeKb - Bioinformatics Knowledgebase

High throughput sequencing (HTS) technologies are capable of sequencing multiple DNA molecules in parallel, enabling hundreds of millions of DNA molecules to be sequenced at a time. High-throughput sequencing (HTS) is a newly invented technology. Although it is more expensive, but HTS can be applied to non-model organisms. HTS is fast, cheaper than traditional DNA sequencing methods, and generates a massive amount of data. But at the same time, HTS has a high error rate, and as HTS generates sequence fragments only.


Features

  • Rapidly sequence whole genomes

  • Zoom in to deeply sequence target regions

  • Utilize RNA sequencing (RNA-Seq) to discover novel RNA variants and splice sites, or quantify mRNAs for gene expression analysis

  • Analyze epigenetic factors such as genome-wide DNA methylation and DNA-protein interactions

  • Sequence cancer samples to study rare somatic variants, tumor subclones, and more

  • Study the human microbiome and discover novel pathogens

Next generation methods of DNA sequencing have three general steps:


LIBRARY PREPARATION

Firstly, DNA is fragmented either enzymatically or by sonication (excitation using ultrasound) to create smaller strands. Adaptors (short, double-stranded pieces of synthetic DNA) are then ligated to these fragments with the help of DNA ligase, an enzyme that joins DNA strands. The adaptors permit the sequence to become bound to a complementary counterpart.


Adaptors are synthesized so that one end is 'sticky' while the other is 'blunt' (non-cohesive) with the view to joining the blunt end to the blunt ended DNA. This could lead to the potential problem of base pairing between molecules and therefore dimer formation. To prevent this, the chemical structure of DNA is utilized, since ligation takes place between the 3′-OH and 5′-P ends. By removing the phosphate from the sticky end of the adaptor and therefore creating a 5′-OH end instead, the DNA ligase is unable to form a bridge between the two terminals.


AMPLIFICATION

Library amplification is required so that the received signal from the sequencer is strong enough to be detected accurately. With enzymatic amplification, phenomena such as 'biasing' and 'duplication' can occur leading to preferential amplification of some library fragments. Instead, there are many types of amplification process which use PCR to create large numbers of DNA clusters.


Emulsion PCR

Emulsion oil, beads, PCR mix and the library DNA are mixed to form an emulsion which leads to the formation of micro wells.


Bridge PCR

The surface of the flow cell is densely coated with primers that are complementary to the primers attached to the DNA library fragments. The DNA is then attached to the surface of the cell at random where it is exposed to reagents for polymerase based extension. On addition of nucleotides and enzymes, the free ends of the single strands of DNA attach themselves to the surface of the cell through complementary primers, creating bridged structures. Enzymes then interact with the bridges to make them double stranded, so that when the denaturation occurs, two single stranded DNA fragments are attached to the surface in close proximity. Repetition of this process leads to clonal clusters of localized identical strands. In order to optimize cluster density, concentrations of reagents must be monitored very closely to remain away from overcrowding.


SEQUENCING

Several competing methods of Next Generation Sequencing have been developed by different companies. Their names are enlisted here;

  • 454 Pyrosequencing

  • Ion torrent semiconductor sequencing

  • Sequencing by ligation (SOLiD)

  • Reversible terminator sequencing (Illumina)

  • 3′-O-blocked reversible terminators

  • 3′-unblocked reversible terminators

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