Keywords

1 Introduction

Deoxyribonucleic acid (DNA), a fundamental part of the central dogma of molecular biology, defines the complete organism. After the discovery of DNA in 1953 by two eminent scientists, sequencing its genes for studying the genotype and phenotype of cells was necessary. Frederick Sanger and Coulson in 1975 came up with the method of sequencing DNA (Sanger and Coulson 1975), and due to its drawbacks 2 years later, they developed another method based on chain termination of nucleotide strand (Sanger et al. 1977). It is considered to be the first-generation sequencing technology to be commercialized with a wide range of applications in molecular cloning, breeding, and evolution. In spite of its accuracy and applicability, traditional Sanger sequencer did not find scope because of its high cost and low throughput per run (Yadav et al. 2014). Molecular biologists from every nook of the world wanted to develop a faster, high-throughput, cheaper, and accurate method to sequence DNA.

Next-generation sequencing (NGS) technologies fulfilled the needs of emerging molecular biology laboratories. NGS technologies sequence thousands to millions of strands at the same time (Hall 2007). Roche 454 sequencer emerged as the first NGS platform working under the principle of pyrosequencing (Ronaghi et al. 1996). Following Roche, Illumina and ABI SOLiD commercialized their genome analyzer. HeliScope and PacBio RS sequencer developed by Helicos BioSciences and Pacific BioSciences respectively eliminated the PCR amplification step and consumed the total run time (Harris et al. 2007). Although these platforms utilized fluorophores and optical imaging to detect the sequence reads, Ion Personal Genome Machine of Life Technologies use pH detection system to accurately predict the sequences (Niedringhaus et al. 2011). Read length of the NGS technologies increased remarkably as a cause of nanopore detection system with greater accuracy and short run time. Due to high read length, sequencing larger fragments or even the whole genome is now possible with some required modifications. In spite of the remarkable success with biological nanopores, its inherent drawback such as nanopore instability at high temperatures was noted (Kang et al. 2005). To ameliorate the nanopore technology sustainable for upcoming generation, synthetic solid-state nanopores are under progress for commercialization, hoping that these nanopores will devote effective genetic analysis.

In this chapter, we will be discussing in brief on individual sequencing topics commencing with traditional Sanger sequencing, followed by an interconnection with current NGS technologies, and finally concluding with emerging NGS method. Merits and demerits of different DNA sequencing approaches along with their necessary bioinformatics tools are also included.

2 Traditional Method

2.1 Sanger Sequencing

Sanger and Coulson in 1975 described their first method known as “plus and minus” for sequencing DNA (Sanger and Coulson 1975), but due to its ineffectiveness Sanger and his co-workers in 1977 launched another sequencing method known as the “chain termination method” that uses chain-terminating inhibitors (Sanger et al. 1977). Since then, DNA sequencing has been carried out in the Sanger sequencer using the chain termination approach as a key principle. Prior to sequencing, the fragmented DNA strands are cloned into a suitable bacterial vector (having high copy number) for in vivo amplification. Then, sequencing starts with amplified template denaturation, annealing of primer to the resulting ssDNA, and extension of annealed primer. After incorporation of fluorescently labeled dideoxynucleotides (ddNTPs), each round of primer extension is randomly terminated. In any given fragment, each fluorescent label on the terminating ddNTP corresponds to a particular nucleotide (either A/T/G/C). Fragments are then separated based on their molecular weight by high-resolution electrophoresis separation method carried out in a capillary polymer gel. A detector coupled to the capillary chamber excites the fluorescent labels of the separated fragments by laser (Fig. 1). The resulting four-channel emission spectra provide the readout also known as “trace,” and these traces are then translated by base-calling software into DNA sequences (Ewing and Green 1998; Shendure and Ji 2008).

Fig. 1
figure 1

Sanger’s chain termination sequencing workflow. (a) Fragmentation of DNA into small fragments. (b) Cloning and amplification of fragments into suitable vector. (c) Cycles of DNA sequencing by chain termination and synthesis of ddNTP terminated, labeled products ladder. (d) Capillary electrophoresis detection to generate sequencing readout/trace

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3 Current Methods

3.1 Roche 454 Pyrosequencing

Roche 454 sequencing system (http://www.454.com) was the first NGS technology to be commercialized. In 2005, Jonathan Rothberg (the founder of 454 Life Sciences) launched his first commercial NGS platform in the name of GS 20, and later in 2007 Roche Applied Science acquired 454 Life Sciences and marketed the second version of the 454 instrument as GS FLX (Voelkerding et al. 2009). The working principle of this high-throughput sequencer lies in the collaboration of two approaches, namely, single-molecule emulsion PCR (Tawfik and Griffiths 1998) with pyrosequencing technology (developed by Nyren et al. 1993 and refined by Ronaghi et al. 1996, 1998). Pyrosequencing is a DNA “sequencing-by-synthesis” method where synthesis/release of a pyrophosphate takes place during nucleotide incorporation (Ronaghi et al. 1996). A picotiter plate (PTP) is a microfabricated fiber-optic slide in which the pyrosequencing reaction takes place. One PTP measures about 44 μm in diameter and consists of more than a million reaction wells per plate with each well having 3.4 × 106 picoliter scale holding capacity. The walls of the wells are metal coated to ameliorate the signal-to-noise discrimination, and a charged couple device (CCD) camera is placed opposite to the PTP that records the light emitted from each bead (Margulies et al. 2005; Voelkerding et al. 2009).

A set of reactions occurring in the 454 pyrosequencing system includes fragmentation of the template DNA to prepare a library of several hundred base pairs in length and ligation of the library to 454-specific adaptor oligonucleotides. Next, the library is diluted to single-molecule concentration, followed by their denaturation into single strands and hybridization to Sepharose or Styrofoam beads carrying oligonucleotides complementary to the sequences of the adaptor. The fragment-containing beads (micelles) are emulsified in a “water-in-oil” emulsion mixture with PCR amplification reagents to create individual micelles. Then these isolated micelles are amplified using emulsion PCR (emPCR) to clonally expand individual DNA fragments into 1 million copies on the surface of each bead. Once beads are recovered after amplification, they are individually arrayed by centrifugation into a PTP with sequencing enzymes, and the plate is loaded into GS FLX sequencer. Enzyme ATP sulfurylase, DNA polymerase, luciferase, luciferin, adenosine 5′ phosphosulfate (APS), and apyrase are involved in catalyzing the pyrosequencing reaction (Mardis 2008a, b; Froehlich et al. 2010). During sequencing, the PTP acts as a flow cell that allows the flow of dinucleotide triphosphates (dNTPs) into the wells.

The enzyme DNA polymerase incorporate these nucleotides as a complementary base to the template strand with the release of inorganic pyrophosphate (PPi). The unmatched bases are degraded by the apyrase enzyme. ATP sulfurylase converts the released PPi into ATP, which then drives the luciferase reporter enzyme to signal luciferin to use the ATP to generate light (Fig. 2). This localized luminescence is transmitted through the PTP and gets recorded on a CCD camera. The amount of light generated is directly proportional to the number of nucleotide incorporated. With the repetitive cycles of pyrosequencing, wells of PTP are subsequently imaged, the signal-to-noise ratio is analyzed and filtered, and algorithmically (Newbler software) a translated output in the form of a linear sequence is given (Ronaghi et al. 1996, 1998; Voelkerding et al. 2009).

Fig. 2
figure 2

454 Pyrosequencing workflow. (a) Emulsification of beads containing DNA fragments ligated to 454-specific adapters, amplification of micelles by emulsion PCR, and emulsion is broken to recover the beads. (b) Loading of beads into the picotiter plate (PTP). (c) Stacking of beads and pyrosequencing enzymes in the PTP. (d) Illustration of pyrosequencing reaction that occurs in a PTP

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3.2 Illumina Sequencing

A highly parallelized adapted version of traditional Sanger sequencing is the Illumina sequencing that works on the principle of sequencing-by-synthesis (Illumina 2010). Initially, this platform originated from the work of Turcatti and colleagues from Manteia Predictive Medicine, Switzerland, and later it merged with four companies Solexa (Essex, UK), Lynx Therapeutics (Hayward, CA, USA), Manteia Predictive Medicine, and Illumina, respectively (Shendure and Ji 2008). Library is constructed by fragmentation of DNA template into 200–300 bp in length, and two different adaptors are ligated to both ends of the strands using PCR. The HiSeq 2500 sequencer uses a solid surface (known as single-molecule array or flow cell) which is an optically transparent slide with eight individual lanes and sequences complement to the adaptor attached on it (Bentley et al. 2008). Fragments are denatured into single strands, and with the help of flanked adaptor, they are annealed to the flow cell complementary sites. The free end adaptor of the array-bound DNA bends down and hybridizes to an opposite adaptor thereby creating a bridge. The bridged DNA strand that acts as a template for the synthesis of its complementary strand is cloned by using fluorophore-unlabeled nucleotides, and hence this process is known as bridge amplification. After amplification, strands are denatured and cleaved. The flow cell will now contain more than 40 million clusters wherein each cluster is composed of approximately 1000 clonal copies of individual template strands (Morozova and Marra 2008). During sequencing reaction, all four dNTPs along with DNA polymerase are added simultaneously to the channels of the flow cell. Each dNTP is bound to a unique fluorescent label reversible dye terminators. Upon nucleotide incorporation, the reversible terminator is cleaved, and the process is repeated until the template strand is made (Fig. 3). A colored fluorescent signal is generated once after the incorporation of each nucleotide, which is then recorded by an imaging device to determine the sequence (Sucher et al. 2012).

Fig. 3
figure 3

Illumina sequencing workflow. (a) Hybridization of the adapter-ligated ssDNA to the flow cell. (b) Bridge amplification to generate clonally amplified clusters. (c) Denatured and cleaved strands are sequenced. (d) Polymerase incorporates nucleotides and releases the fluorophore to begin the next synthesis cycle

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3.3 SOLiD Sequencing

Supported oligonucleotide ligation and detection (SOLiD) platform was originated from George Church laboratory in 2005 and later commercialized by Applied Biosystems (Life Technologies) (Shendure et al. 2005). The technology lies in the principle of sequencing by ligation of oligonucleotides to the DNA template. A mixture of short DNA fragments from the library are ligated to oligonucleotide adapters, immobilized on the surface of 1 μm paramagnetic beads, and clonally amplified by emulsion PCR (Dressman et al. 2003). Emulsion is then broken, and beads are recovered and are attached covalently to the surface of specially treated glass slide that is placed into a fluidics cassette within the sequencer to generate dense, disordered array. In this platform, two slides are processed per run, one slide receiving sequencing reactants, and second slide is being imaged (Mardis 2008b). The ligation-based sequencing begins with the annealing of universal primer (n) complementary to the array of amplicon-bearing beads that provides a 5′ phosphate substrate for DNA ligase. After annealing, DNA ligase is added along with four semi-degenerate octamer/8-mer fluorescent oligonucleotides in an automated manner within the instrument. The 8-mer fluorescent oligonucleotide is a probe that contains 2 probe-specific bases consisting of one of 16 possible 2-base combinations (TT, GT, and so on), 6 degenerate bases (nnnzzz), a ligation site (on first base), a cleavage site (on fifth base), and 4 different fluorescent dyes (linked to the last base) (Liu et al. 2012). Adjacent to the universal primer (n), DNA ligase seals the phosphate backbone when a matching 8-mer probe hybridizes to the DNA template sequence. A fluorescent readout is followed identifying the ligated 8-mer probe, which corresponds to one of the 16 possible combinations. Subsequently, linkage between the fifth and sixth base of the 8-mer is chemically cleaved removing the fluorescent group allowing further ligation. Seven ligation cycles (rounds) are performed to extend the first primer (n) and then the synthesized strand is denatured from the adapter/template. The second round of sequencing is initiated with hybridization of a new n − 1-positioned universal primer (that is offset by 1 base in the adapter sequence) to the synthesized strand and continues the rounds of ligation-mediated sequencing (Fig. 4). Process is repeated each time with a new primer with a successive offset (n − 2, n − 3, and so on). In this approach, all the templates are sequenced twice (Voelkerding et al. 2009; Shokralla et al. 2012). Finally, 2-base-calling processing software decodes the fluorescence generated from the universal primers into sequence reads.

Fig. 4
figure 4

SOLiD sequencing workflow. (a) Octamer interrogation probes with 2 probe-specific bases on the left and 16 possible 2-base combinations on the right. (b) (1) In the first round of sequencing, adapter (P1)-linked template is annealed to the primer (n), and the primer is interrogated by a probe having 2 specific bases (e.g., here AT) complementary to the template. (2) Fluorescence emitted during probe annealing and ligation is recorded, and the last three degenerative bases are cleaved. (3) Annealing and ligation of next probe and so on. (4) and (5) The primer (n) is fully extended by seven cycles of ligation and the resulting product is denatured from the template for second round of sequencing with a new primer (n − 1) and vice versa

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3.4 True Single-Molecule Sequencing (tSMS)

True single-molecule DNA sequencing method was first developed by Stephen Quake and colleagues in 2003 and commercialized in 2007 by Helicos BioSciences (Cambridge, MA) as HeliScope sequencer (Braslavsky et al. 2003). It is a unique platform utilizing sequencing-by-synthesis approach without any clonal amplification of the DNA template by PCR (Harris et al. 2007; Pushkarev et al. 2009). Library is prepared by fragmenting template DNA into small, 100–200 bp fragments and polyadenylation of generated DNA fragments with poly-(A) tail at 3′ end, with the final adenosine fluorescently labeled with Cy3 (Shendure and Ji 2008). Denatured polyadenylated template strands hybridized to the poly-(T) oligonucleotides are immobilized on the surface of flow cell at a capture density of up to 100 × 106 template strands/cm2. The instrument records the position of each fluorescently labeled template on the array prior to sequencing and expects the sequence read in that position. After the positional coordinates are recorded by highly sensitive CCD camera, label is cleaved and washed before sequencing. Sequencing cycle begins by adding DNA polymerase and one of four Cy5-labeled (Cyanine-5) dNTPs to the flow cell resulting in template-dependent extension of DNA strands (Fig. 5). Here sequencing is asynchronous, i.e., during a particular round of sequencing, all the templates will not incorporate a nucleotide (Voelkerding et al. 2009; Anderson and Schrijver 2010). The emitted fluorescence signal is captured, and the images are recorded using CCD camera to determine incorporated nucleotide. After imaging, the label is chemically cleaved and removed by washing, and the cycle is repeated with the next Cy5-labeled dNTP. Therefore this sequencing platform utilizes single DNA molecules as templates rather than clonally amplified clusters and results in a higher sequencing output per run (Deschamps and Campbell 2010).

Fig. 5
figure 5

True single-molecule sequencing (tSMS) workflow. (a) Clockwise illustration of four steps involved in tSMS: (1) Dye-labeled nucleotide is incubated on the flow cell for incorporation. (2) The unincorporated, labeled nucleotide molecules are removed by washing. (3) Then the incorporated Cy-5-labeled nucleotide is imaged by highly sensitive CCD camera. (4) Finally, the dye label is cleaved from the incorporated nucleotide, and the next sequencing cycle follows

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3.5 Single-Molecule Real-Time (SMRT) Sequencing

Pacific BioSciences (Menlo Park, CA) commercially introduced PacBio RS sequencer in 2010 for single-molecule real-time (SMRT) sequencing. It is a real-time, fluorescent single-molecule sequencing platform that relies on sequencing-by-synthesis approach (Korlach et al. 2010). A dense array nanostructure called as zero-mode waveguide (ZMW) manufactured on chip surface is the chief component of the sequencer. The chip is fabricated by perforating a thin metal film supported by a transparent substrate and contains more than 10–1000 ZMW well, with each well measuring about 10–50 nm in diameter. ZMW is a light-focusing structure that allows for real-time observation of DNA polymerization. Adapters are ligated onto the ends of the template DNA fragments, and a primer complementary to the adapter sequence is annealed. A calculated ratio of primer-annealed DNA templates and DNA polymerase molecules mixture is supplied to the instrument by a diffusion-mediated process. DNA polymerase is engineered to have a decreased rate of polymerization and a unique ability to incorporate fluorescently modified nucleotides (Mardis 2013). Next, fluorescently labeled nucleotides (linked to the phosphate) are added sequentially to the wells, and DNA polymerase incorporates these nucleotides to create DNA strand complementary to the template. The optical system is finely tuned to measure extremely small detection volume (20 × 10−21 L) of fluorescence emitted during reaction (Anderson and Schrijver 2010). Once phosphate diester bond is formed, a fluorescent signal is produced corresponding to the each incorporated nucleotide which is detected by the optical system (Eid et al. 2009). After each incorporation and detection event, fluorophore is cleaved from the growing strand and diffuses out of the wells (Fig. 6). The sequencer incorporates nucleotides at the rate of 10 bases/s, giving rise to a chain of a thousand nucleotides within minutes (Korlach et al. 2008). The instrument monitors all the wells constantly during the run, performs numerous calculations, condenses the data, and analyzes it to produce sequence reads.

Fig. 6
figure 6

Single-molecule real-time (SMRT) sequencing ZMW chip and workflow. (a) DNA polymerase bound ssDNA template is immobilized at the bottom of a zero-mode waveguide (ZMW) nanostructure illuminated by laser light at the bottom. Fluorescently labeled nucleotides are added for sequencing the bound template. (b) Cyclic event of dinucleotide triphosphate (dNTP) incorporation in a ZMW with detected fluorescence intensity graph. (1) Attachment of phospholinked nucleotide to the template by ZMW bound polymerase. (2) This creates an elevation of fluorescence output on the corresponding color channel. (3) The fluorescence dye linked to the nucleotide pyrophosphate is cleaved after forming the phosphodiester bond with the template, and then it diffuses out of the ZMW ending the fluorescence pulse. (4) and (5) Next nucleotide binds the active site as the polymerase translocates and subsequent pulse is generated

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3.6 Ion Torrent Semiconductor Sequencing

Ion Personal Genome Machine (PGM) is a fast, simple, massively scalable, versatile, and less costly sequencing technology commercialized in 2010 by Ion Torrent technology, a company that was later acquired by Life Technologies during the same year (Pareek et al. 2011). The sequencer relies on the principle detection of hydrogen ion concentration that is released as a by-product during nucleotide incorporation into the template by DNA polymerase (Rothberg et al. 2011). Library fragments constructed by DNA fragmentation and adapter ligation are hybridized to the bead containing adapter complementary sequences and then amplified by emulsion PCR (emPCR). After successful amplification, emulsion is broken, and the resulting beads are enriched. Enriched beads are annealed to the sequencing primer and are deposited into the highly dense microwells of an Ion Chip along with DNA polymerase by mild centrifugation. Ion Chip is a specialized silicon chip designed to detect pH changes of individual microwells (Mardis 2013). Upper surface of the chip delivers the required sequencing reactants. Beneath the microwells, an ion-sensitive layer followed by a field-effect transistor (FET)/ion sensor sub-layer is aligned to measure changes in pH of the solution (Niedringhaus et al. 2011). Sequentially nucleotides flow into the wells, wherein each well acts an individual DNA polymerization reaction chamber. Nucleotide complements with the template DNA and gets incorporated by polymerase action releasing hydrogen ions (H+) (Fig. 7). As a result, FET detects the change in pH of the solution (ΔpH), and a potential change (ΔV) is recorded as a direct measure of nucleotide incorporation events (Hui 2014). For instance, if two identical bases are incorporated, the voltage is double, and two identical bases are recorded. Next, if an unmatched nucleotide enters the well, no voltage change is recorded, and no base is called. The sequencer directly converts the received chemical information into digital information by processing the signals and by using base-calling algorithms to produce the DNA sequence associated with individual reads (http://www.lifetechnologies.com/).

Fig. 7
figure 7

Ion Chip and Ion Torrent semiconductor sequencing workflow. (a) Ion Torrent and Ion Chip are used for sensing pH changes that occur during sequencing. (b) and (c) pH-based sequencing reaction in which protons released when nucleotide is incorporated into the template by polymerase

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4 Emerging Method

4.1 Nanopore Sequencing

Nanopore sequencing technology emerged from the independent work of David Deamer and his colleagues at the University of California (Santa Cruz, CA, USA) in 1996 (Branton et al. 2008). It is a fast-growing real-time sequencing technology, in that sequencing is carried out in a very thin porous membrane with pores ranging in nanoscale size (nanopores). Current is applied across the nanopore, and the negatively charged ssDNA traverses through it toward the positive terminal causing a change in electrical conductivity across the nanopore. The potential difference generated in picoamperes (pA) is measured using a circuit (Gupta 2008). This technology obviates the need to amplify the template fragments by PCR, synchronous washing of the reagent, and synthesis of strand complementary to the template and to reduce time (Ku and Roukos 2013). Based on the kind of nanopore, the technology is categorized into two types, namely, biological nanopore sequencing and solid-state nanopore sequencing.

4.1.1 Biological Nanopore Sequencing

In this type of sequencing, α-hemolysin (αHL), a protein nanopore, is held between a phospholipid bilayer separating two chambers filled with KCl solution. Two electrodes are placed on the opposite sides of the bilayer, and an electrical potential is applied during sequencing. The amount of current passing through the nanopore in a given moment varies depending on the shape, size, and length of the nucleotide that blocks the ion flow through the pore (Hui 2014). An exonuclease enzyme is attached to the nanopore surface to cleave individual nucleotide molecules from the DNA as it enters. Negatively charged ssDNA translocates through the membrane as positive potential is applied to the electrode. Translocation velocity varies with different parameters such as electric potential, type of nanopore, and strands of DNA (whether ss/ds) (Steinbock and Radenovic 2015). Ionic current is partially blocked during translocation producing different magnitudes of current disruption that in turn aids in differentiation among the four nucleotides (Fig. 8a).

Fig. 8
figure 8

Nanopore sequencing technology. (a) Nanopore sequencing reaction involves unwinding of dsDNA by φ29 DNA polymerase and controlled translocation of unwounded ssDNA through MspA. (b) Biological nanopores: (1) heptameric α-hemolysin (αHL) nanopore and its dimensions and (2) Mycobacterium smegmatis porin A (MspA) nanopore and its dimensions. (c) Solid-state nanopore

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Specificity of nucleotide detection across the membrane is tenfold higher in current MspA (Mycobacterium smegmatis porin A protein) nanopore than classical αHL (Manrao et al. 2011) (Fig. 8b). Translocation velocity of DNA can be slowed down for improving detection accuracies by combining MspA with DNA polymerase φ29. Oxford Nanopore Technologies (ONT) is only the leading company that develops, commercializes, and updates these types of bio-nanopore-based sequencing platforms. ONT in collaboration with Professor Daniel Branton, George Church, and Jene Golovchenko of Harvard, David Deamer and Mark Akeson of UCSC, and John Kasianowicz of NIST commercialized their first developer version of the nanopore sequencer “MinION” on 2014 as an early access program (MAP or MinION Access Program) to the researchers and investigators. MinION is a portable device commercialized for the analysis of DNA and RNA in real time. Recently, in May 2015, ONT introduced the second version of the device “MinION” as “MinION MkI” featuring its improvements in performance and easy access. Improvements in library preparation kits and temperature controlling system, an increase in total number of nanopores per flow cell, etc., can be found in MinION MkI. PromethION, a small benchtop platform developed by ONT for high-throughput real-time analysis of the DNA and RNA samples, is currently under the scheme of commercialization (Oxford Nanopore Technologies, UK).

4.1.2 Solid-State Nanopore Sequencing

This type of sequencing uses nanopores fabricated mechanically on a solid-state material such as silicon nitrides, silicon oxide or metal oxides, and graphene (Fig. 8c). These artificial nanopores are considered to be more stable than biological nanopores as it eliminates membrane instability and protein positioning drawbacks. IBM in collaboration with 454 Life Sciences is developing artificial nanopores by drilling a 10 nm titanium nitride membrane, which are separated by insulating layers of silica. When DNA strand translocates through the nanopore, the electric field across the layer is flipped resulting in immobilization of the strand and significant measuring of ionic current. Graphene is another new single-atom membrane that is 1–5 nm thick and has pores with a diameter of 5–10 nm (Niedringhaus et al. 2011). Graphene membrane improves sequencing and detection accuracies by allowing the passage of one base at a time (Pennisi 2012). Therefore, these kinds of nanopores are robust in their unique ability and leads researchers toward the next-generation sequencing.

5 Bioinformatics Tools

Sequence reads generated from various NGS platforms are analyzed using commercially available bioinformatics tools (Table 1). Each tool has a unique function, categorized as (Lee et al. 2013):

  1. 1.

    Read alignment based on the reference genomes.

  2. 2.

    De novo assembly involves grouping of generated short reads into contigs which are further assembled into scaffolds to reconstruct original genomic DNA of a new species.

  3. 3.

    Identification of single nucleotide genetic variant (i.e., mutation) in the assembled sequence reads.

  4. 4.

    Detection of functional variants altering the protein coding regions of individual genomes.

  5. 5.

    Detection of large alterations such as indels, inversions, and translocation in the DNA (~1 kb–3 Mb).

  6. 6.

    Transcriptome assembly.

Table 1 Bioinformatics tools for analyzing the raw data generated from various NGS platforms
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6 Comparison of Various NGS Platforms

See Table 2.

Table 2 Comparison of various NGS platforms and their advantages and disadvantages
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7 Conclusion and Perspective

Next-generation sequencing (NGS) technologies contribute rapid, high-throughput, cost-effective probing of genomes of individual organisms. Geneticists have seen a prompt inclination in the sequencing technologies over the past decade. Despite the merits and limitations of each platform, these platforms can be used in tandem for decoding the entire genome of a distinct organism. Modern NGS technologies serve prominent roles in scrutinizing genome-level mutation and gene expression to divulge phenotype of diverse strains. Therefore, these technologies have augmented our minds toward the immense knowledge about the organism and their active systems at molecular level. In the successive years of NGS technologies, progress in state-of-the-art bioinformatics software will be of critical importance to elicit sophisticated amount of generated sequenced reads.