Demand for fast, inexpensive sequencing methods was fulfilled by discovering “high throughput DNA sequencing” methods, i.e. Next-generation sequencing method, which played a crucial role in revolutionizing genomic research. In this era of advancement, many other sequencing methods have emerged, but NGS is still rigorously used by research for sequencing. I have discussed why NGS is still popular among scientists in “Benefits of NGS over first and third-generation sequencing”; here, in this blog, we will discuss the principle and types of sequencers used in the NGS technique.
Principle and steps of NGS
NGS or high throughput sequencing is an innovation consisting of various technologies that can be best described as Massive parallel DNA sequencers, capable of sequencing large no of DNA in a single reaction. Although various machines are developed which use different techniques to sequence DNA, they also share some common steps like sample preparation, sequencing machine and Data analysis.
Sample preparation
The library is the collection of randomly sized DNA fragments representing the sample input. The different applications of NGS like Whole-genome sequencing (WGS), Exome sequencing (Exome-seq), RNA Sequencing (RNA-Seq) and Methylation Sequencing (Methyl-seq) influence library preparation. Despite the difference in the source, such as genomic DNA, reverse-transcribed RNA or cDNA, and immunoprecipitated DNA, Exome sequences, regulatory elements, for sequencing, dsDNA template is generated that follow some of the precise common library preparation steps before the commencement of the sequencing.
Firstly, the DNA is cut into numerous smaller sequence-able fragments (by sonication or digestive enzymes like DNase I), followed by repairing overhangs into blunt ends. The size of fragments depends upon the type of sequencing application and desired insert size (referring to the library portion between the adapter sequences). The trimming or end repair is done to prevent dimerization. Subsequently, A-tail formed with the addition of Adenine at the ends of fragments allowing the adaptor with thymine overhang to base pair. Furthermore, The short double-stranded piece of synthetic DNA (Adaptors) is covalently ligated by ligase enzymes. These adaptors serve as a primer for downstream amplification and a medium for the attachment to the flow cell. Depending upon the NGS technique, the library is directly used for sequence or use different amplification methods. Namely, two types of PCR, i.e. emulsion PCR and bridge PCR, are used for amplification.
Types of NGS sequencing machines
Sequencing by synthesis (Illumina sequencing technology)
This technique developed by Shankar Balasubramanian and David Klenerman at the University of Cambridge is based on Sanger sequencing but, distinct from it, as it does not use the chain termination method. In this technique, step-by-step, all four types of fluorescently labelled reversible terminator dNTPs are added to the sequencing chip simultaneously. Upon incorporating one of the four dNTPs on the growing DNA copy strand, the polymerization is terminated which and the rest of the dNTPs are washed off. The specific color fluorescent signal of each nucleotide is read by a camera at each cluster and recorded. Further, the fluorescent molecule and terminal group are cleaved and washed away, and following another set of all four modified dNTPs are added, leading to the sequencing of template DNA. This sequencing reaction repeated simultaneously on a huge number (millions) of different template molecules spread out on the sequencing chip.
Pyrosequencing (454 sequencing )
It is based on the principle of ‘sequencing by synthesis’ in which sequencing is carried out by detecting nucleotide incorporation by DNA polymerase. In pyrosequencing, incorporating dNTPs on the DNA template releases the pyrophosphate, which can be converted into ATP following the reaction.
PPi + APS (adenosine 5-phosphosulfate) ATP-sulfurylase ATP + Sulphate
This ATP generated can be combined with luciferin and O2 and catalysed by luciferase to generate detectable light, which is read and recorded.
ATP + luciferin + O2 luciferase AMP + PPi + oxyluciferin + CO2 + light
To know which nucleotide is added to an immobilized (on bead: emulsion PCR) growing strand of DNA, the reaction is carried out in the flow cell (controlled by computer), where only one nucleotide is added at a time. Thus, if a particular, e.g. Adenine, is added and incorporated, a light will be generated with the release of PPi, which acts as a cue for incorporation and thus recorded. However, if the incorporation does not take place, no detectable light will be detected generated. If the identical two nucleotides are added subsequent, then the intensity of light will be high.
Ion semiconductor sequencing (Ion Torrent sequencing)
This technique is based on ‘Sequencing by synthesis’ but, instead of labelled dNTPs, it uses a semiconductor transistor, which detects the release of H+ ions during sequencing with nucleotide incorporation into a strand of DNA by the polymerase. Each cluster of DNA is directly located above a semiconductor transistor capable of detecting change pH generated by the release of H+ ions during the incorporation of dNTPs. This change in pH detected converted and recorded in the form of electrical signals. Like Pyrosequencing, this whole sequencing reaction is carried out in a flow cell, and a computer controls release on nucleotides.
Sequencing by Ligation (SOLiD DNA sequencing)
This is the only sequencing method that does not use DNA polymerase; instead, it is based on ‘Polony sequencing’ (mixture of word Polymerase and colony) and uses DNA ligase enzyme to identify the nucleotide present at a given position in the DNA sequence. Each fragment of DNA is bound to beads and amplified with PCR. The 16 8-mere nucleotide probe has two actual dinucleotide bases; 3 universal bases which bind to any of the four nucleotides; and three universal bases with fluorescent dye. The 16 possible dinucleotide permutation corresponds to a dye colour (red, green, blue or yellow/orange).
The process initiated with the annealing of primer corresponding to the P1 adaptor, next to which an appropriate 8-mere fluorescently labelled probe hybridizes to the DNA template. Upon hybridization, it is ligated to the primer sequence through a DNA ligase. This probe is guided by two dinucleotides specific to the bases of the DNA template. Unbound oligonucleotides are washed away, and the fluorescent signal is recorded. Once the cue is detected last 3 universal nucleotides carrying florescent are cleaved off; further, the next cycle commences. This cycle of ligation is repeated approximately seven times, after which DNA is denatured, and another sequencing primer offset by one base from the previous primer is used to repeat the steps. In total, five sequencing primers are used to complete the sequencing of each fragment.
Upon sequencing by any suitable methods, the data collected are analyzed using various bioinformatics tools, which will be discussed in another blog.
Reference
Head, S. R., Komori, H. K., LaMere, S. A., Whisenant, T., Van Nieuwerburgh, F., Salomon, D. R., & Ordoukhanian, P. (2014). Library construction for next-generation sequencing: overviews and challenges. Biotechniques, 56(2), 61-77.
Rizzo, J. M., & Buck, M. J. (2012). Key principles and clinical applications of “next-generation” DNA sequencing. Cancer prevention research, 5(7), 887-900.
Rothberg, J. M., Hinz, W., Rearick, T. M., Schultz, J., Mileski, W., Davey, M., … & Bustillo, J. (2011). An integrated semiconductor device enabling non-optical genome sequencing. Nature, 475(7356), 348-352.
https://en.wikipedia.org/wiki/Pyrosequencing