DNA sequencing technologies

The advent of sequencing techniques has played an important role in the study of organism genomic sequences. Tedious were early attempts to sequence DNA; 12 bases of coherent ends of bacteriophage were calculated through the usage of key extension methods. Maxam and Gilbert identified 24 bases of the binding site for lactose-repressors by copying them into RNA and sequencing them. Two methods Sanger, Maxam-Gilbert was more common earlier and still in use to determine the order of the nucleotide bases adenine , guanine, cytosine , and thymine in a molecule of DNA. Although with the advances of technology these days, much of the digital instrument is focused on these. The sequencing technologies are divided into three separate sequencing groups.

First Generation of Sequencing

Sequencing of Sanger and Maxam-Gilbert has been listed as First Generation Sequencing Technologies. Similar sequencing methods have been developed in many ways; but only tiny DNA fragments (less than one kilobase) may be sequenced.

Sanger sequencing

Sanger sequencing sometimes referred to as the process of chain termination or the process of dideoxynucleotide, or synthesis sequencing. It consists of using a single strand of the double-stranded DNA as a sequencing template. This sequencing is done with chemical nucleotides, called dideoxy-nucleotides. These nucleotides, once inserted into the DNA chain, are used as dNTP for nucleotide elongation, inhibit further elongation, and the elongation is complete. Thereafter, DNA fragments finished with dNTP are collected with varying sizes.The fragments are differentiated by their scale using a gel block, so an imaging device may be visualized with the subsequent bands corresponding to DNA fragments.

For a few decades the Sanger sequencing has been widely used for single or low-throughput DNA sequencing even today. However, the pace of processing that doesn’t require the replication of the diverse genome is challenging to further develop.

Maxam-Gilbert Sequencing

Maxam-Gilbert depends on chemical cleaving of nucleotides, which is most successful with tiny polymers of nucleotides. Chemical therapy brings in splits in each reaction at a limited proportion of one or two of the four nucleotide bases. This reaction results in a collection of identified particles, which can be differentiated by electrophoresis according to their scale. Sequencing by Maxam & Gilbert included either double or single-stranded DNA molecules that were labelled at one end of a 32P chain. In each of four reactions, chemical treatment created breaks at a small proportion of one or two of the four nucleotide bases. Thus, a sequence of named fragments in each molecule were created, from the radiolabeled end to the first cut spot. The fragments were then measured by gel-electrophoresis and authoradiography was done, resulting in an illustration of bands referring to the radiolabeled fragments from which the series was deduced.

Second Generation Sequencing

The first-generation sequencing was dominant for three decades, especially Sanger sequencing; however, the cost and time was a significant issue. In 2005 the emergence of a new generation of sequencers, break the limitations of first-generation sequencers. The essential characteristics of second-generation sequencing technology are:

  1. The generation of many millions of short reads in parallel
  2. The speedup of sequencing the process compared to the first generation
  3. The low cost of sequencing and
  4. The sequencing output is directly detected without the need for electrophoresis
  5. Shotgun sequencing of randomLy fragmented genomic DNA without the need of cloning via a foreign host cell
  6. Library amplification is performed on a solid surface or beads within miniature emulsion droplets

Short read sequencing methods separated into two side approaches: ligation sequencing, and synthesis sequencing. Because a high-throughput sequencing is readily feasible, sequencing of the second generation is also known as Next-Generation Sequencing ( NGS). Different platforms based next-generation sequencing are 454, ABI SOLiD, Illumina, and Semiconductor sequencing (Ion Torrent).

Roche/454 Sequencing

In 2005, a company named 454 Life Sciences Corporation took the first steps in the NGS movement. The name 454 derived from this business which was later purchased by Roche in 2007. The Concept behind 454 is focused on pyrosequencing, which was a Pyrosequencing Ab approved technology. This method depended on the inorganic pyrophosphate (PPi) being produced during PCR when a complementary base was inserted. The sequencing reaction happens very quickly, in the range of milliseconds, and a charging-couple-device ( CCD) camera may detect the light generated.The use of a picotiter plate allowed hundreds of thousands of reactions to take place in parallel and was able to generate reads for around 500-600 base pairs long for the millions or so of wells that would be required to contain sufficiently clonally-coated bead. The 454 technological breakthrough miniaturized the sequencing reaction, enabling simultaneous sequencing reactions to occur in a limited space utilizing a smaller number of reagents. The newer version of the 454 instruments will produce reads up to 1000 bp in lengths, generating millions of reads per cycle.

454 Pyrosequencing Method (Low and Tammi, 2017)

Description of Figure:

  1. 454 Pyrosequencing, procedure initiated with a single-stranded library with adaptors ligated on both ends.
  2. The adaptor binds to the beads, which is further subjected to emulsion PCR, which generates millions of copies of a single DNA fragment on each bead.
  3. The beads are placed into PicoTiter Plate for sequencing by detection of base incorporation during PCR.
  4. When a nucleotide base is incorporated, inorganic pyrophosphate (PPi) is generated, which is converted to ATP by sulfurylase and luciferase uses the ATP to oxyluciferin and light.

The 454 ‘s crucial drawback is the difficulties of measuring the real amount of homopolymer tract bases (e.g., TTTTT). It lacked blocking mechanism to avoid the inclusion of several similar bases during elongation of DNA. It was difficult to determine how many bases there are until the homopolymer is longer than eight bases, only more accurate light signals flow meaning. Signals of too large or too low amplitude led to the amount of nucleotide bases being underestimated or overestimated, which triggered irregular nucleotide recognition.


ABI completed the purchase of Agencourt Personal Genomics in 2006, which allowed the launch of novel NGS technologies known as Supported Oligo Ligation Detection (SOLiD). Thermo Fisher Science is actually the proprietor of Strong sequencing technologies. Because of the di-base encoding method the Strong sequencing scheme is difficult. The measures of sample preparation before ligation of the probes are quite close to 454 programs. The adaptors are ligated to previously broken DNA and enriched by emulsion PCR on beads. The beads are then connected to a glass slide by means of covalent bonds, with which bases are ligated and identified. To bind to the established adaptor chain, a universal sequence-primer is used.

Overview of the SOLiD sequencing process (Low and Tammi, 2017)

Description of figure:

  1. Each ligation cycle starts with the 8-mer probe binding to the template and then ligated for its detection. Then, cleavage occurs to remove three nucleotides and a tagged dye.
  2. Structure of the 8-mer probe.
  3. An illustration of the sequence determination process during each ligation cycle of the primer rounds. Position 0 is a part of the adaptor sequence, and the template sequence is only revealed from position one onwards.

To reach the required sequence duration the ligation and cleavage cycle may be performed several times. While the SOLiD framework is unusual in that it can store the sequence of oligo color calls to be used for mutation calls, this has raised problems for bioinformatics research as most methods are focused on DNA calls rather than color space models.


Solexa initially released the first sequencer, Genome Analyzer, and was later purchased by Illumina in 2007. Some of Illumina ‘s crucial strengths is its capacity to produce large DNA sequence data output at reduced expense, while generating only short sequences. In addition to the beneficial low-cost high-performance sequencing, Illumina does higher than the 454 homopolymer sequencing method, because it employs reversible terminator sequencing chemistry.

Overview of Illumina Sequencing (Low and Tammi, 2017)

Description of figure:

  1. Genomic DNA was sheared, size selected, and then attached with adaptors at both ends.
  2. DNA library, placed on the flowcell to allow for complementary binding at one end of the adaptor to probes that are coated on the surface. The solid-phase bridge amplification generated clusters of single DNA fragments. Reverse strands cleaved and washed away.
  3. Sequencing initiated with primer addition to the remaining forward strand, further addition with the incorporation of one nucleotide at a time.

Illumina devices come with drawbacks too. The sequence end of 3′ appears to be of poorer quality than the end of 5′ which ensures that any sequences from the end of 3′ will be filtered out because it is beyond a certain defined level. Sequence-specific errors for twisted repeats and GGC sequences have also been identified (Low and Tammi, 2017).

Ion Semiconductor Sequencing

Semiconductor sampling, also known as Ion-Torrent sampling, was originally held by Life Technologies, and then launched by Thermo Fisher Scientific. It was comparable to 454 pyrosequencing technology because, unlike other second-generation techniques, it did not use fluorescent-labelled nucleotides. Furthermore Ion Torrent is going quicker than Illumina.

Similar to the 454 and SOLiD schemes, the Ion Torrent includes the library planning and emulsion PCR phases on the beads. The main distinction lies in identifying the absorption of nucleotides not focused on fluorescence or chemiluminescence, but calculating the H+ ions produced during the cycle instead. Nucleotide identification became possible due to a miniature pH-sensor semiconductor. For DNA elongation every of the four DNA bases was provided sequentially, if the base matched the reference, then a signal was identified.

For the homopolymer area in the signal system should be enhanced, but it was difficult to identify the real amount of bases (more than six bp), which triggered insertion and deletion error at a pace of around ~1 percent. However, many methods have been attempted to address the limitations of the Ion Torrent network, including refinement of the bioinformatics system, and improvement of scope breadth and uniformity.

Third-Generation Sequencing

Despite becoming common, second-generation sequencing typically involves the PCR amplification stage, which is a lengthy execution-time process. Further, second-generation sequencers were unable to tackle the diverse genomes of repeated regions. The third wave of sequencing developed to address the issues created by the second-generation sequencers. The third generation of sequencing will deliver a low cost of sequencing and simple sample preparation without the need for PCR amplification considerably faster than second generation sequencing technologies in an implementation cycle. Two key methods describe sequencing of third generation

  • The single-molecule real-time sequencing approach (SMRT)
  • The synthetic approach that relies on existing short reads technologies.

The most widely used sequencing platforms in third-generation sequencing are MinION and Oxford Nanopore sequencing.

Single-Molecule Real-Time (SMRT) sequencing

Pacific Bioscience, rendered crucial advances that enabled DNA synthesis to be observed in real time. Every nucleotide with phospholinks has a fluorescent dye added to the phosphate line, rather than the nucleus. The phosphate chain is cleaved during DNA elongation, and thus the dye mark diffuses forward. The DNA prototype is able for next nucleotide to embrace. Another main advancement is the usage of the Zero-mode-waveguide (ZMW) as the foundation integration detection tool. Such ZMWs are contained inside an SMRT container. ZMW are tens of nanometers diameter wells microfabricated in a metal film which, in effect, was deposited on a glass substrate surface. Growing ZMW comprises a DNA polymerase connected to the sequencing fragment at the bottom and the goal DNA fragment. The DNA fragment is combined with fluorescent-labeled nucleotides (with various colours) by DNA polymerase during the sequencing reaction. Whenever a nucleotide is inserted, a luminous signal is emitted which is registered by the sensors.

Oxford Nanopore Sequencing

The Oxford Nanopore sequencing has been developed as a tool for evaluating nucleotide order in a sample of DNA. Oxford Nanopore Technologies launched the MinION device in 2014, which could produce longer readings, allowing greater resolution of the structural genomic variations and repeated sequences.

The DNA fragment in nanopore sampling is transferred into a modified nanopore membrane. Once a motor protein connected to the pore passes the DNA fragment across the pore, it produces a change of an ionic current triggered by variations between the passing nucleotides that fill the pore. The ionic current variability is slowly reported on a graphic model, and then interpreted to classify the series. The transcription is performed on the main strand producing the ‘template learning,’ and the parallel strand of DNA attached to the hairpin is learning creating the ‘complement write.’ The resultant consensus series called ‘two-direction read’ or ‘2D’ is produced when the ‘template read’ and ‘complement read’ are merged.

The benefit Oxford Nanopore sequencing provides is its low cost and compact scale. Information is shown on the screen as the sample is loaded without hesitation into the cable.

Helicos sequencing

The Helicos sequencing method was the first industrial application of fluorescent single-molecule sequencing, currently sold by the Helicos Biosciences. Currently the Seqll sequencing company uses the Helicos sequencing method and HeliScope single-molecule sequencers to decode genomic DNA and RNA. DNA is sheared, tailed with polyA, and hybridized to a flow cell surface that includes oligo-dT for sequential sequencing-by-synthesis of billions of molecules. The DNA molecules polyA-tailed fragments are hybridized similarly to the oligo-dT50 attached on the surface of discarded glass flow cells. The introduction of fluorescent nucleotides with a ending nucleotide delays the cyclic cycle until a single nucleotide signal has been identified for each DNA segment, and then the cycle is replicated before the fragments have been sequenced entirely. This sequencing method is a mixture of DNA polymerase sequencing via hybridization and synthesis sequencing. Sample preparation involves no ligation or PCR amplification and therefore generally removes the GC material and size differences found in other technologies. The read lengths of HeliScope sequencing vary from 25 to over 60 bases, with the maximum of 35 bases. This approach successfully sequenced the human genome to provide signatures for the disease in a clinical assessment and sequenced RNA to generate tissue and cell quantitative transcriptomes.


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