Determining the order of the nucleotides present in the DNA is called as a DNA sequencing. Our DNA is made up of the 4 nitrogenous bases adenine, thymine, cytosine and guanine. A long chain of the bases which codes for a particular protein is called as a gene.
For understanding the structure and function of a gene, it is very important to know the sequence of bases present into a particular gene.
DNA sequencing serves this purpose. In a sequential manner, the long chain of DNA is read by the machine.
The present article is huge, in-depth, giant and contains all the information on the DNA sequencing, how to do sequencing and different methods of DNA sequencing. The content of the article is,
- History of DNA sequencing
- What is DNA sequencing
- What are the steps in DNA sequencing
- Different methods of DNA sequencing
- How to sequence DNA
- Applications of DNA sequencing
- Limitations of DNA sequencing
History of DNA sequencing
The story of DNA begins when Watson and Crick discovered the structure of DNA in the year 1953. In 1964, Richard Holley who performed the sequencing of the tRNA was the first attempt of sequencing the nucleic acid.
Using the technique of Holley, Walter Fieser sequenced the genome of bacteriophage MS2 (RNA sequencing). The sequenced molecules were RNA, Yet DNA sequencing was not performed.
In the year 1977, Fredrick Sanger postulated the first method for sequencing the DNA, called a chain termination method.
In the same year, the chemical method of DNA sequencing was developed by Allan Maxam and Walter Gilbert. The genome of bacteriophage X174 was sequenced in the same year using the chemical degradation method.
The chain termination method and the chemical degradation methods of DNA sequencing are not automated, the use of manual preparation made it tedious. The first semi-automated DNA method was developed by Lorey and Smith in the year 1986.
Later on, in the year 1987, Applied biosystem developed a fully automated machine-controlled DNA sequencing method. After the development of the fully automated machines, the era of the 2000s become a golden period for the sequencing platforms.
Another attempt was made by Applied Biosystem in 1996 and developed another sequencing platform called capillary DNA sequencing.
After that, the human genome project was completed by using the combination of these methods in the year 2003.
The fast, accurate, reliable and highly efficient next-generation sequencing platform was developed in the year 2005 by Ssolexa/Illumina. Some of the milestone into the DNA sequencing is shown in the figure below,
What is DNA sequencing?
The DNA is made up of purine and pyrimidine bases. It is a long chain of A, T, G and C. knowing the order in which the nucleotides are arranged on a DNA is called a DNA sequencing. Know more on the structure of DNA: DNA story: The structure and function of DNA.
The DNA sequencing technique is the combination of wet lab and dry lab work, once the reaction is completed into the web lab the data will be processed into the dry lab for computational result analysis.
Broadly, in any of the sequencing platform, the labelled nucleotide is periodically bound to the complementary nucleotide present into the single-stranded target DNA. The signal of labelled nucleotide is recorded by the computer and convert it into the sequence of A, T, G and C.
This is the basic mechanism. The labelled nucleotides may be radiolabeled or fluorescently labelled, depends on the platform what we are using.
What are the steps in DNA sequencing?
Steps mentioned below are the generalized representation of DNA sequencing, it may vary from platform to platform.
- Sample preparation (DNA extraction)
- PCR amplification of target sequence
- Amplicons purification
- Sequencing pre-prep
- DNA Sequencing
- Data analysis
The starting sample for sequencing is DNA. For that, we have to extract DNA from the organism whose DNA we want to sequence. The DNA may be animal DNA, plant DNA, bacteria DNA or plasmid DNA.
However, the quality of DNA is a major concern in DNA sequencing. For that proteinase K DNA extraction method is highly recommended or spin column based DNA extraction method can be more appropriate.
The DNA with the 260/280 ratio of 1.8 is selected for further steps of DNA sequencing. The quantity of DNA may be nearly 100ng or more. Good quality of DNA reduces the chance of failure in the sequencing reaction.
PCR amplification of target sequence:
In case of the sequencing of a particular gene, we have to first amplify that gene for getting multiple copies, the rest of DNA is discarded which is not need in sequencing. Nonetheless, in whole genome sequencing, the entire genome of the organism is sequenced. The whole genome sequencing method is described in the middle part of this article.
For PCR, flanking primers are designed which flanks the region of our interest. The amplification is performed at the desired annealing temperature of primers for 35 cycles, good quality of amplicons will be generated.
After the amplification, the PCR product is run of 2% agarose gel along with the DNA ladder. Once the amplification achieved, the PCR product is proceeding for the purification.
If you check the purity of the PCR product at 260 and 280nm wavelength, it will always be 1.80, exactly 1.80 because the amplicon is the pure DNA fragment. If any contaminants are present in the DNA, it will not be amplified. Then why amplicon purification required?
Unbound primers, primer dimers, unused Taq DNA polymerase, unused DNA templates and other unused PCR reaction buffer components hinder in DNA sequencing. That is why amplicon purification is required.
Here, the alcohol purification cannot work, we have to purify our PCR product with the spin column PCR amplicon purification kit.
Sample preparation is needed before DNA sequencing. For that, both the end of the PCR amplicon is ligated with the known DNA adapters.
The known sequence-specific primers are added to the reaction with the sequencing mastermix. The sequencing mastermix contains the fluorescent or labelled nucleotides which is detected by the detector.
Along with that, Taq DNA polymerase and other PCR enhancers are added that enhance the amplification.
Now the sample tube placed into the DNA sequencer. Here first, the PCR product (which is now our sample for sequencing) is denatured into the heat govern step and becomes single stranded. The high fidelity Taq DNA polymerase adds the labelled nucleotide to the growing DNA strand.
That signals of the addition of each complementary nucleotides are recorded by the machine and the data is sent the computer.
Once the whole DNA is sequenced, the result is saved into one unique file formate. The inbuilt software (provided by the manufacturer) processed the data and compared it with the available data.
The sequence which we read is compared with the sequence data of that particular gene, available online. Any alteration such as SNP and other smaller copy number variation is detected by the software and indicated into the end result.
Different methods of DNA sequencing:
- Maxam and Gilbert method
- Chain termination method
- semiautomated method
- automated method
- Whole genome shotgun sequencing method
- Clone by the clone sequencing method
- Next generation sequencing method
Terminologies used into the article:
|Fragment library||A collection of the entire strand of the DNA (to be sequenced) fragments.|
|Gaps||Here unsequenced region of the DNA is called the gap|
|Conting||A continuous sequence of the DNA assembled from the single|
|Read||The output data came from the sequencer machine to the computer for one particular sequence.|
|Coverage||The number of the times the sequencing machine covered the DNA sequence.|
The Maxam and Gilbert method was developed into the year 1977 also called chemical cleavage method. By using this method, they had sequenced 24 nucleotides. However, their method is published after two years of sangers method.
“The single-stranded DNA is cleaved at the specific location with the help of the chemicals at specific base location and the fragments of DNA is then run on polyacrylamide gel electrophoresis.”
Obviously, DNA extraction is the very first step. After that, the DNA is denatured using the heat denaturation method and single-stranded DNA is generated.
The phosphate (5’ P) end of the DNA is removed and labelled by the radiolabeled P32. The enzyme named phosphatase removes the phosphate from the DNA and simultaneously, the kinase adds the 32P to the 5’ end of the DNA.
4 different chemicals are used to cleave DNA at four different positions; hydrazine and hydrazine NaCl are selectively attack pyrimidine nucleotides while dimethyl sulphate and piperidine are attacking purine nucleotides.
- Hydrazine: T + C
- Hydrazine NaCl: C
- Dimethyl sulphate: A + G
- Piperidine: G
An equal volume of 4 different ssDNA sample is taken into the 4 different tubes each containing 4 different chemicals. The samples are incubated for sometimes and electrophoresed on polyacrylamide gel electrophoresis.
The results of the chemicals cleavage of four different tubes are shown in the figure below.
The separated DNA fragments are detected using the autoradiographic analysis. Due to the radiolabelled 32P end of the DNA, the DNA bands are visualised into the autoradiography.
At our glance:
The method is more accurate than Sanger sequencing. The method is best suitable for DNA footprinting and DNA structural studies. The main achievement of the chemical degradation method is the use of the purified DNA directly.
Even in the present times, the Maxam Gilbert method is applicable in DNA fingerprinting and genetic engineering studies.
Still, The scalability of the method is very low; it is restricted up to 400bp. Furthermore, the use of radiolabelled molecules and harmful chemicals makes the method more tedious for routine use.
Sanger and co-workers developed a chain termination method of DNA sequencing, after some time of the Maxam and Gilberts method. The method is also called as first generation method of DNA sequencing because of its easy setup and handling.
The chain termination method is also referred to as a dideoxynucleotide sequencing because of the use of the special types of ddNTPs. The ddNTPs are different from normal dNTPs. The ddNTPs contains the hydrogen groups instead of a hydroxyl group in the dNTPs.
Because of that, the phosphodiester bonds cannot be formed between adjacent nucleotide and addition of nucleotides are stopped there. Thus, the DNA chain formation is terminated by the addition of the ddNTPs.
The process of Sanger sequencing is broadly divided into 3 steps:
- DNA extraction: using any of the DNA extraction protocols
- PCR amplification: using the flanking primers, dNTPs, ddNTPs, Taq DNA polymerase and PCR buffer.
- Identification of the amplified fragments: using autoradiography, PAGE or capillary gel electrophoresis.
Read more on DNA extraction methods: Different types of DNA extraction methods
The process of chain termination is started with the DNA extraction and purification. DNA extraction can be achieved using the proteinase K method or the phenol-chloroform DNA extraction method. Or we can use ready to use silica-column based kit method.
The main aim of any DNA extraction method is to achieve the purity nearby ~1.8 and the quantity over 100ng.
In the next step, the PCR am amplification is performed by designing four different reactions
|Reaction “A”||Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers||Labeled ddATPs|
|Reaction “G”||Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers||Labeled ddGTPs|
|Reaction “T”||Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers||Labeled ddTTPs|
|Reaction “C”||Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR buffer, primers||Labeled ddCTPs|
Each tube contains the same amount of the PCR reagents but in each tube, extra ddNTPs are added as shown into the table.
The flanking primers are such a primer that binds to the region near to our sequence of interest or the sequence what we are going to read.
Now in the next step, the Taq DNA polymerase adds the dNPTs.
The Taq DNA polymerase is a normal polymerase use into the PCR, it expands the growing DNA strand by addition of the ddNTPs. Interestingly, once it adds the ddNTP instead of dNTP the chain expansion is stopped or we can say terminated.
The termination process is complete in 4 different tubes for 4 different ddNTPs. For example, in the ddATP tube, it terminates the chain at all the position where the adenine is going to bind.
The amplified PCR products are loaded on to the polyacrylamide gel electrophoresis. The DNA fragments migrate into the gel based on the size of the fragments. The smaller fragments run faster towards the positive charge than the larger fragments.
Before the PAGE run, the amplified DNA fragments are further denatured by heat. Depending upon the types of labelling the gel is then analysed under UV light or X-ray film. The banding pattern of the amplified product is shown in the figure below,
The Sanger sequencing is the gold standard method for research as well as in the diagnosis in recent days because of the easy setup and high reproducibility. It is commercialised and semi-automated.
Instead of reading the sequence on the PAGE (manually), the sequence is read computationally with the help of the detectors. Here the laser beam of light passes through the sample, based on the different fluorescent lights emitted by different ddNPTs the signal is sent to the computer.
The computer generates the real-time absorbance peak of the different fluorescent and gives the final results in the form of the fluorescent peaks. The typical results of the Sanger sequencing are shown in the figure below,
How to interpret the PAGE result for Sanger sequencing?
Now take a look at the figure above,
The smaller fragments of DNA can pass easily from the gel pores and hence can migrate faster than the larger fragments. So we have to start readying our sequence from the bottom because the fragment at the bottom is the smaller.
Therefore, at the bottom the fragment in the in the tube ddCTPs is the smallest, Now start sequencing at this point. Our first nucleotide is “C”. Before doing sequencing, count all the bands in the gel and compare it with the length of your sequence. if your sequence length is 16, then 16 bands must be present into the gel.
Read your sequence as indicated in the figure.
Automated DNA sequencing
The Sanger method was tedious, however, recent advancement into the sequencing makes it easy and rapid to use. The semi-automated Sanger sequencing method is based on the principle of Sanger’s method with some minor variations.
Instead of the 4 different reactions, the automated DNA sequencing carried out in the single tube. Which means on a gel the DNA runs in a single lane.
The four different types of the fluorescent-labelled oligo primers are used rather than the simple primers. This four different fluoro labelled primers give four different colour picks with four differents ddNPTs (instead of Fluor labelled primers, fluorescently labelled ddNPTs can also be used).
The PAGE method is not capable for separating of all the fragments in the single reaction. Therefore, alternatively, the capillary gel electrophoresis method is used. This method separates each and every single fragment preciously.
The four different coloured peaks are generated due to the four different fluorescent labelled primers.
By labelling the primer or the dNTPs, the machine reads the sequence accurately, on a single lane capillary electrophoresis. We can sequence more than 300 samples in a single run on an automated DNA sequencing platform.
The capillary electrophoresis used to separate DNA molecules on the basis of the size, it can even separate a single basepair change. The chromatogram generated through the C.E is sent output as a fluorescent peak.
The advanced semi-automated Sanger sequencing method is more accurate, reliable and faster than the traditional method.
What happened inside the tube?
At our Glance:
The read capacity of the Sanger sequencing is higher as compared with the chemical degradation method. It can sequence 700 to 800bp in a single run, therefore, it is more suitable for sequencing of the genomes of bacteria and other prokaryotes.
It is more advanced and automation is possible for Sanger method. Even the error rate is very low as compared with the chain termination method.
Still, it is time-consuming and a high-cost method.
Fact: depending upon the time required on sanger sequencing, more than 50 years required to sequence all the bases present in human DNA through conventional Sanger sequencing.
In 1993, Bertil Pettersson, Mathias Uhlen and Pål Nyren described the pyrosequencing method.
The method is based on the detection of the pyrophosphate, released during the chain reaction of nucleotide addition. Here the order of the nucleotide is determined by the PPi released during the joining of two adjacent nucleotides (3’OH- 5’P).
In contrast with other methods, instead of a single polymerase, two additional enzymes are required in the pyrosequencing method. The three enzymes are:
- DNA polymerase (without exonuclease activity)
All three enzymes work in a sequential manner for the detection of the PPi. The real-time polymerase activity monitoring allows the detection of the released pyrophosphate in a cascade of the enzymatic reaction,
(DNA)n + dNTP ———————— (DNA)n+1 + PPi (Polymerase)
Addition of one dNTP removes one pyrophosphate from the DNA.
PPi + APS —————————– ATP + SO4-2 (ATP sulfurylase)
ATP + luciferin + O2 ——————— AMP + PPI + oxyluciferin + CO2 + photon (luciferase)
Here the reaction is completed into the three steps:
The enzyme polymerase adds the dNTPs to the single-stranded DNA. if the correct complementary base is added, the pyrophosphate released.
The enzyme sulfurylase converts the PPi into the ATP (energy) with the help of the APS (adenosine 5´ phosphosulfate).
The ATP act as a substrate for the luciferase activity (more specifically “firefly luciferase”). With the help of the ATP substrate, the luciferase converts the luciferin into the oxyluciferin in the presence of oxygen and the photon of light is released.
Once the correct nucleotide is added, the amount of the light released by the enzymatic reaction is detected by the charged device coupled camera, photodiode or photomultiplier tube. This is the basic fundamental of the pyrosequencing set up.
Based on the substrate used into the technique two types of pyrosequencing methods are available: solid phase pyroseq and liquid phase pyroseq.
At our Glance:
The pyrosequencing method is more accurate than the Sanger sequencing which has the capacity to add nucleotides up to 500 nucleotides. The major advantage of the pyrosequencing is the speed of the reaction.
However, the method required more chemical steps than a chain termination method which makes it more complex. Furthermore, the read length is too short as compared with the automated sequencing.
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Whole genome shotgun sequencing
The whole genome shotgun sequencing is another modification of the chain termination method, thus Sanger sequencing. The method is developed to sequence the entire genome of an organism rather than a single gene.
The principle of the shotgun is the same as sanger, one additional step of DNA fragmentation allows to read multiple fragments.
The entire genome of an organism is fragmented with the help of endonuclease enzymes or by the mechanical techniques. After that, the smaller fragments of DNA sequenced individually into the machine.
The computer-based software analyses each and every overlapping fragment and reassembled it to generate the complete sequence of the entire genome.
The method can be divided into four steps:
- Fragmentation of a DNA: with the help of restriction endonucleases or physical method
- Formation of libraries of the subfragments: the fragments are ligated into the vectors and an entire library for the different vector are generated
- Sequencing the subfragments: each library is sequenced individually.
- Generation and reading the contigs: the overlapping fragments called as contigs are read by the computer.
The fragments generated by the lysis may around 2 to 20kb.
Importantly, the shotgun sequencing reads both the sequences (it sequence the double-stranded DNA) based on that contigs data, it identifies the gaps remained unsequenced.
Whole genome shotgun sequencing method.
Do you know?
The shotgun sequencing concept was originally discovered by the Fred Sanger and his colleagues for sequencing the whole genome.
At our glance:
The shotgun sequencing is faster and cheaper than the previous techniques.
The technique becomes more aggressive if any reference sequence is available to align (it can find out the gaps and mutations as fast as possible).
Gene or chromosomal mapping and tedious enzymatic reaction steps are not required in the shotgun sequencing method.
It has the power to sequence the whole genome of an organism (more time required to sequence the complex genome such as humans and animal).
The major portion of the human genome is made up of non-functional repetitive DNA. it is very difficult for shotgun sequencing to assembled the repetitive DNA sequences. And it is nearly impossible if any reference genome is not available.
The technique solely depends on the computer. A huge, powerful, supercomputer is required to work efficiently.
The gap cannot be further filled by the techniques.
The technique becomes feeble if reference genome sequence data is not available.
“In the year 1981, The genome of cauliflower mosaic virus was sequenced by shotgun sequencing method.”
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Clone by clone sequencing
Clone by clone sequencing is another method for sequencing the whole genome. This method is one of the pioneers and traditional methods used during the Human genome project.
The method is somehow the same as the shotgun sequencing with one or two additional steps.
Instead of smaller fragments, very large fragments of DNA are generated, the location of the fragments are mapped on the chromosomes.
Multiple copies of these fragments are generated by inserting it into the BACs (bacterial artificial chromosome).
In the next step, the inserted fragments are broken into smaller fragments and further inserted into the vectors. The library of the vector is prepared for sequencing.
The sequencing is performed as like the shotgun and the overlapping fragments are assembled by the computer.
Interestingly, in the last step, based on the data of the chromosome map (which we are generated earlier) is used to assemble the sequences. Individual sequences can be arranged based on their chromosomal locations.
At our Glance:
Any gaps in sequencing can be identified easily because the fragment of the DNA is taken from the known locations.
The different scientist can work on different chromosomes because of the mapping. A large number of DNA can be sequenced at once.
However, the method is very tedious because additional steps such as mapping and cloning are involved.
Chromosomal mapping takes more time and manpower which makes this technique time consuming and costlier.
Cloning the chromosomal part such as telomeres and centromeres are difficult to achieve which makes this technique even more toughest.
“During the year, 1980 and 1990, the genomes of the nematode worm, C. elegans and the yeast, S. cerevisiae was sequenced using the clone by clone sequencing, respectively.”
Nonetheless, the clone by clone technique is the part of the history of the Genetics because it was involved in the human genome sequencing.
Actually, the method shotgun sequencing was evolved from the clone by clone sequencing technique.
Every platform, we discussed here, had some drawbacks and limitations. Any sequencing platform is available which is fast, accurate, reliable, accurate and cost-effective? The answer is “yes”.
The next-gen sequencing, the next level of revolution in the sequencing technology.
The next generation sequencing platform is different from the sanger or chain termination method of DNA sequencing. Broadly, it amplifies millions of copy of a particular fragment in a massively parallel fashion and the reads are analysed by the computational program.
The NGS process is quite complex, However, it can be divided into the 4 different steps:
- Library preparation
- Cluster generation
- DNA sequencing
- Data analysis
1. Library preparation:
The library preparation is the combination of two reactions viz, fragmentation and ligation. The extracted DNA or the cDNA is fragmented with the help of the restriction digestion and the end of the small fragmented DNA is ligated with the known DNA sequences.
The known DNA sequences are called as the adapters and the process is called as adapter ligation. Once the adapters are ligated, the library of the smaller DNA fragments is generated.
The unbounded DNA fragments are washed by the washing buffer, the process of library preparation is called as tagmentation.
2. Cluster generation:
What is the role of adapters??
The short oligo sequences of nucleotide are immobilised on the solid surface which is complementary to our adapter sequences.
Once the library of our fragmented DNA is loaded into the cell, it is bound with the immobilised oligos in the solid surface and by the bridge amplification, the cluster of the DNA sequence are generated.
Here, in the bridge amplification, the DNA fragments bend over and bind to the next oligo which creates a bridge. A primer binds to this DNA sequence and amplified vertically.
Two new single-stranded DNA is generated by bridge amplification.
As the polymerase adds the nucleotide into the bridge amplification, the signal for the addition of the nucleotide is recorded each time. This will generate the multiple sequencing databases for the, particularly amplified fragment.
4. Data analysis:
The read generated by the sequencing can be aligned to the reference genome sequence and by doing this we can identify any addition, deletion or variation into the sequence.
At our glance:
The NGS is the most advanced, fast, accurate and 100% effective technique in modern day science.
These are the common types of sequencing platform used in the genomic labs. Besides this, several other sequencing methods are:
- Single molecule real time (RNAP) sequencing
- Single molecule SMRT(TM) sequencing
- Helioscope(TM) single molecule sequencing
- DNA nanoball sequencing
- SOLiD sequencing
- Illumina (Solexa) sequencing
- Polony sequencing
- massively parallel signature sequencing (MPSS)
- High throughput sequencing
Application of DNA sequencing
In medical science, DNA sequencing can be used in the identification of the genes responsible for hereditary disorders. New mutation can also be detected with the help of the DNA sequencing
In forensic science, it is used for parental verification, criminal investigation and identification of individuals through any of the available samples such as hair, nail, blood or any tissue.
In the agriculture industries, identification of GMO species can be possible with the help of the DNA sequencing methods. Any of the minor variation into the plant genome can be identified with the help of the DNA sequencing.
It is used to construct maps such as whole chromosomal maps, restriction digestion maps and genome maps.
Identification of open reading frames or non-open reading frames can be possible by sequencing. It can also identify the protein-coding sequences.
DNA sequencing is used in exon/ intron, repeat sequence and tandem repeat identification and detection.
Also, gene manipulation and gene editing studies and identification of new variations are only possible by the sequencing techniques.
Metagenomic studies are nowadays possible by sequencing methods such as pyrosequencing.
It is further used in the Microbial identification and study of the new bacterial species. The sequencing technique advances the microbial identification, eliminating the traditional and time consuming culturing methods.
Nowadays the microbial identification and characterisation become more rapid and accurate by using sequencing. By comparing the sequence of the target microbes with the available data, the scientist can identify new mutations and new strains.
The sequencing techniques specifically, the NGS has a great application in the oncology and cancer detection. So many mutations in different genes which are responsible for a particular type of cancer can be identified using sequencing. The NGS facilitate to study each and every gene involve in the particular types of cancer. Diagnosis of cancer is facilitated by the advancement in the NGS.
Sequencing facilitates evolutionary studies by comparing the gene of different organisms and can create the evolutionary maps of an organism. One can also identify any changes present in the DNA sequence from evolution.
Prior to disease development, data can also be generated by studying the asymptomatic high-risk population of particular disorder through the sequencing method. Preventive actions can be taken for genetic disorders.
Do you Know?
Sir Shankar Balasubramanian is the only known Indian scientist who was involved in the development of a sequencing platform. He was the principal investigator in the development of Solexa/ Illumina Next generation sequencing.
Limitations of DNA sequencing
A sequencing platform is a computer algorithm based assistive technique which relies on the data processing of the computer. For that, a huge, high-speed supercomputer required. Also, repeated sequences, tandem sequences, massively fragmented genes, gaps in the sequences, duplicated segments and error in the pre-processing of the sample are some of the key factors limit the sequencing techniques.
Methods such as NGS can become gold stander in the diagnosis of multigenic disorders. Also, it can be a useful tool in preimplantation genetics and prenatal diagnosis, in future. Additionally, it can be a game changer in the personalised medicines and in in vitro studies of gene editing. Advancements are required to decrease the cost, time duration and error by increasing the specificity, reliability and accuracy of the sequencing methods.