The sequencing of DNA molecules started with the development of chemical cleavage method of Maxam-Gilbert during 1970s followed by di-deoxy chain termination method. However, the whole genome sequencing of the any organism was difficult and time consuming task, hence leads to search for other alternatives. This has resulted in emergence of numerous sequencing methodologies, which can sequence about 2.8x106 base pairs/ day. Most of the modern technologies used for sequencing are modifications of Sanger’s method. Ronaghi et al. developed pyrosequencing, a simple technique analysing short to medium length DNA sequences. It can be used to analyse genetic variations like single-nucleotide polymorphisms (SNPs), determination of short sequence repeats (SSRs) and allelic imbalance in RNA, DNA methylation status and assessment of gene copy number. It is based on the principle “Sequencing by synthesis”. It is able to characterize diverse sequence populations with detection of low frequency variations efficiently. Highly sensitive mutational analysis like discovery of unknown mutations, quantification of alleles in mixed populations, characterization of contiguous and multivariable mutations can be performed. Its precision and accuracy provided with increased discriminatory power and sensitivity is pre-requisite of forensic analysis. This technique involves a cascade of enzymatic reactions; into which new strand is synthesized by addition of nucleotides and after each addition one pyrophosphate (PPi) is released. Because of the release of PPi, the reaction is called pyrosequencing. After each addition of nucleotide to the growing end, one PPi is released and visible light is generated, which is proportional to the number of incorporated nucleotides during synthesis of its complementary strand enzymatically.
The pyrosequencing completes in four stages: (i) amplification of target DNA by emulsion PCR. In order to prepare DNA for sequencing, first genomic DNA is sheared mechanically or enzymatically and short oligonucleotide adapters are linked to the template DNA. To increase the copy number of target DNA, it is amplified in emulsion PCR, instead of cloning it in bacteriophage vector. In emulsion PCR, on the surface of agarose beads, short oligonucleotide primers, which have sequence complimentary to adapter sequence, is attached. Water in oil emulsion is prepared and concentration of template DNA is kept so that each micelle contains only one template strand. This micelle also contains the Bst polymerase and dNTPs. Thermal cycling of this water and oil micelle is called emulsion PCR. Breaking of this micelle releases millions of copy of template DNA, ready to be sequenced. (ii) Bead containing template on its surface is deposited in the picotiter microwell (iii) Immobilized enzymes (ATP sulphurylase, lucifearse, apyrase and DNA polymerase) are also deposited and subjected to thermal cycling (iv) the pyrosequencing reaction takes place, in which, complementary strand is synthesized using enzymes and other substrates present in reaction mixture, It result in pyrogram, a collection of signal peaks, corresponding to the nucleotide incorporated. For detection of pathogen from clinical samples, the technique is rapid and reliable. It is having important role during diagnosis, treatment and prophylactic measurements. Considering the various uses of pyrosequencing, in present article the technique is discussed in detail with elaborating its uses.
Pyrosequencing technique is based on sequencing-by-synthesis principle. The method is based on the sequencing of single stranded DNA and now-a-days sequencing of double stranded DNA is also possible. The detection system is based on finding of released PPi during the strand synthesis. The primer is hybridized with single-stranded DNA template and mixed with adenosine -5’- phosphosulphate (APS), luciferin and the enzymes; DNA polymerase, ATP sulfurylase, luciferase and apyrase. Sequentially the solutions of A, C, G and T nucleotides are added and removed by apyrase.
During the polymerization step, one nucleotide is added by DNA polymerase and simultaneously PPi is released in a ratio proportional to the incorporated nucleotide. The PPi released is now converted to ATP by ATP sulfurylase enzyme in the presence of APS. The resulting ATP provides the energy and drives the luciferase enzyme mediated luciferin to oxyluciferin conversion, and visible light is detected. This light signal may be recorded using a photomultiplier or charge coupled device (CCD) camera. The detected signal is observed as a peak of signal in the pyrogram and the peak is analogous to electropherogram in dideoxy sequencing. Apyrase enzyme cleaves remaining ATP and non-incorporated dNTPs from the reaction mixture. Usually a time lapse of 65 seconds is kept between a nucleotide dispension Apyrase enzyme is added to remove unused dNTP without in between washing step. The enzyme is strong catalyst and even in low quantities, it efficiently degrades the unincorporated nucleosides triphosphates to diphosphates and further diphosphates to nucleoside monophosphate. Apyrase is resistant to accumulation of end products in comparison to other nucleotide-degrading enzymes. During pyrosequencing, detection of false signals also occurs, when dATP was added into the pyrosequencing solution since dATP is a substrate for luciferase. The problem was resolved by addition of dATPáS instead of dATP in the polymerization reaction. dATPáS, being inert for luciferase, can be incorporated efficiently by all DNA polymerases tested leading to successful use of the strategy for sequencing of PCR-generated DNA material.
Pyrosequencing modus operandi
1. Preparation of DNA template
Solid phase template preparation and enzymatic template preparation are the methodologies used for generating primed DNA template for pyrosequencing. Streptavidin-coated magnetic beads are used to prepare primed DNA template in solid-phase template preparation. Template preparation by such system provides superior quality sequence data with less background signals. The template may be further purified by ethanol precipitation followed by alkali denaturation to yield ssDNA. Exonuclease I and a nucleotidedegrading enzyme are used in enzymatic template preparation method. Postamplification exonuclease I degrades the unused PCR primers and nucleotidedegrading enzyme removes the nucleotides. Enzymatic template preparation method is used to sequence double-stranded DNA template.
2. Polymerization step
The synthesis of new strand includes the hybridization of DNA template with primer, which is further extended by polymerase enzyme. The nucleotides dATP, dTTP, dCTP, dGTP are added sequentially, if the added nucleotide is complimentary to the base in template strand, it is incorporated in the growing strand and PPi is released.
3. Enzymology of pyrosequencing
The activity of following enzymes is crucial for the precision of this DNA sequencing technology:
(i) DNA polymerase: In pyrosequencing,generally Bst DNA Polymerase, obtained from Bacillus stearothermophilus containing the 5´ ? 3´ polymerase activitybut lacking 5´ ? 3´ exonuclease activity is used for incorporating the nucleotides in complimentary strand
(ii) ATP sulfurylase: The enzyme is memberof transferase family. ATP Sulfurylase catalyses formation of ATP from adenosine 5'-phosphosulphate (APS) and pyrophosphate (PPi).
(iii) Luciferase: Firefly luciferase is a euglobulin protein and it induces oxygenation of luciferin to yield oxyluciferin using molecular oxygen and ATP. It is a two step process; in first step luciferin is adenylated and luciferase acts as an oxygenase on adenylluciferin complex producing oxyluciferin and carbon dioxide. The resultant, oxyluciferin is a unstable, excited compound producing photon of light upon reaching to its ground state. The amount of generated light is equal to the proportion of ATP generated.
(iv) Apyrase enzyme continuously degrade unincorporated nucleotides and ATP
The light generated through enzyme coupled reaction, is captured and recorded and converted to electrical signal by a charge coupled device (CCD) camera and each signal is depicted as a peak in electric pyrogram. A computer generated software couple the signal and the nucleotide addition at particular moment and thus the signals are converted to a nucleotide sequence. The height of each light signal is proportional to the number of nucleotides incorporated. During recent years, deoxyadenosine alfa-thio triphosphate (dáATPS) is substituted for the natural deoxyadenosine triphosphate (dATP). dáATPS, instead of dATP is used as it does not serve as energy for luciferase enzyme and also used by polymerase efficiently. As the sequencing process proceeds, signal peaks are generated in program. The ascending slope of curve shows the activities of DNA polymerase and ATP sulfurylase, the height indicates the activity of luciferase and decending slope exhibits the nucleotide degradation.
Applications of pyrosequnecing
For detailed study and characterization of any organism, it is essential to elucidate the nucleotide sequence of its genome. Pyrosequencing has opened up new avenues for performing sequence-based DNA analysis. Since it has proven its efficacy in surveillance of emerging drug-resistant pathogens of human and animal origin. Currently this method is widely used in many fields such as classifying single nucleotide polymorphism, identification of bacteria and viruses, fungal and viral typing, difficult secondary DNA structure sequence determination, mutation detection, clone scrutiny and DNA/RNA methylation analysis.
a) DNA methylation analysis: DNA methylation is considered as an important epigenetic modification. A methyl group is added to cytosine and adenine nucleotides, where it repress the gene transcription and also associated with several important cellular processes like genomic imprinting, Xchromosome inactivation, aging and carcinogenesis. Anomalous methylation of specific genes has been directly linked with the activation of proto-oncogene, tumor response to chemotherapy and patient survival. Thus, a quantitative measurement of DNA methylation can give estimate about the magnitude of developmental and pathological disorders. Among eukaryotes, methylation generally takes place at CpG island, where cytosine is immediately followed by guanine residue.
Quantification of methylation can be done in this CpG island area. DNA methylation pattern may be linked to the tumor type and abnormal gene expression pattern and cellular response to demethylating agents. Assessment of methylation pattern changes, with regards to tumorigenesis, genetic imprinting, and exposure to environmental toxins may provide an insight for developing therapeutics against methylation associated disorders. DNA methylation is also found to be associated with alteration in organism behavior. A DNA methylation level at the Dopamine Receptor D4 (DRD4) gene is statistically associated with personality and behavioral traits in both humans and non-human animals. Such epigenetic traits are considered to control the behavior. Variation in methylation level is detected to bring behavioral changes in Great tit (Parus major) birds by pyrosequencing technique.
b) Study of microbial community: At present thousands of organisms have been sequenced. The sequence elucidate about their virulence, metabolism, mode of infection and potential targets for antimicrobial agents as well. Pyrosequencing technique has emerged out as an excellent diagnostic tool for detection and identification of microbial community. Generally for detection purpose, DNA markers present in both the conserved as well as variable regions are targeted. The bacterial strains are usually identified by sequencing the 16S rRNA gene. The 16S rRNA gene (16S rDNA) is having highly conserved regions flanked by variable and species specific sequences, signature to one specific organism. 16S rRNA sequencing is also used to distinguish pathogenic bacteria from commensals to saprophytic bacteria, present in the same habitat and identification of contaminating bacteria in industrial waste water systems. The technique is also used for typing fungal isolates, obtained from clinical cases of immune-compromised patients. On the basis of observation of sequencing data, it was found that 18 to 32 bases are sufficient for discriminate between Candida tropicalis, C. albicans, C. krusei, C. parapsilosis, C. glabrata, and Aspergillus spp . A combination of multiplex real-time PCR and pyrosequencing detected the presence of Bacillus anthracis in milk, apple juice, and bottled water by targeting pXO1 and pXO2 plasmids and genomic DNA. Barcoded pyrosequencing for 16S rDNA has helped in providing the insight into the presence of bacterial communities in faecal microbiota of diarrhea affected population.
c) Detection of viral pathogens: Molecular biology is largely dependent on sequencing techniques for a variety of application like determination of genetic variance within and among populations, pathogen testing and typing and determination of resistance. Since pyrosequencing is a robust, accurate, quantitative, and cost effective technique, providing real time data, increasingly it is becoming a standard laboratory technique. Several diagnostics tests have been developed for detection of important animal/bird’s diseases like rabies, new castle diseases, avian influenza etc. Rabies is a fatal zoonotic disease caused by rabies virus, a member of Rhabdoviridae family and Lyssavirus genus. Sometimes rabies related lyssavirus also cause endemic in human population; therefore, it is pre-requisite to develop a reliable molecular detection module for differential diagnosis of rabies from the rabies related lyssaviruses. The target may be achieved by targeting the 3’ terminus of the nucleoprotein (N) gene developed for rapid characterization of lyssaviruses in pyrosequencing protocol. For detecting molecular markers responsible for resistance to M2 ion-channel blockers and for NA inhibitors in influenza A H5N1, H7N9 viruses Pyrosequencing may be used. Its combination with RT-PCR techniques, provides rapid, reliable, high-throughput and cost-effective screening of NA inhibitor-resistant influenza A viruses of subtype H1N1, H3N2 and H5N1 (35). Due to the increasing diversity among H5N1 viruses due to antigenic shift and drift, pyrosequencing RT-PCR is a powerful tool to distinguish viruses belonging to the different HA clades. The same may be achieved by amplifying the specifically variable region of the HA gene. New castle disease causes respiratory and neurological ailment in birds and lead to substantial economic losses. NDV viruses are different in pathogensity, and hence only detection in any specimen is not sufficient to provide exact information regarding the pathogen, hence assessment of virus pathotype is required. Pyrosequencing based Real time PCR has been developed for rapid and reliable detection and pathotyping of NDV virus. The results obtained from pyrosequencing are high quality, rapid and cost-efficient and generate ample sequencing data in comparison to the classical procedures based on chain termination method. These qualities make pyrosequencing a dependable choice as diagnostic tool for NDV virus.
d) Resequencing: Those genes, which are associated with mutations, can be resequenced and resequencing for scanning of mutations can be important application of pyroseqencing. Resequencing gives longer read length than de novo sequencing because here the supply of nucleotide may be specified as per previously generated program and mutation at specific site may be reconfirmed. The same strategy has been used for resequencing of the p53 tumor suppressor gene, where mutations in the gene were effectively determined and quantified. In resequencing, for sequencing a homopolymeric region, a single nucleotide may be added several times as per the signal of previous pyrogram.
e) Genetic testing of polymorphism: Flexibility in primer placement in the pyrosequencing offers an opportunity to assess virtually all genetic markers. All sequence information like short tandem repeats, insertion and deletions, SNPs, and variable gene copy number can be deduced in a single run.
Advantages of pyrosequencing over Sanger’s method
Pyrosequencing is an alternative to the conventional Sanger method. It was first developed at the KTH Royal Institute of Technology, Sweden. Its several advantages like rapidity, reliability, flexibility, ease-of-us and real time data visualization makes it a favoured research tool. In pyrosequencing, the fluorescence signals are converted into nucleotide sequence and there is no requirement of manual reading as in case of Sanger’s method of sequencing there is requirement of reading electropherogram. It is faster and cheaper technique where DNA is sequenced by method generally called as “sequencing by synthesis”. It avoids the used of expensive and hazardous incorporation of labeled primers or labeled nucleotides. Unlike Sanger sequencing, where a gap of at least 20-30 bases from the sequencing primer occurs, pyrosequencing generates the data immediately downstream the sequencing primer. Sample preparation process is relatively fast and consumes only 15 minutes opposing to Sanger sequencing, where it may take upto 4 h including lengthy time periods for PCR cleanup, cyclic amplification and dye cleanup. The greatest advantage is that pyrosequencing generate hundreds of thousands of sequence reads in a single run generating larger magnitude of data.
454 GS FLX Titanium pyrosequencing based system was developed having capacity to read up to 700 bp with 99.9% accuracy. Now-adays it is possible to obtain up to 14 gigabase data per run, with the advantage of completion of run within 10 hours. Pyrosequencing technique avoids the bias inherent to the cloning procedure and directly the DNA can be sequenced without further steps of genomic shearing followed by cloning and subsequent procedures. In the pyrosequencing method, using the barcoding multiplex approach, in which unique sequences are incorporated into the primers and barcoded amplicons are generated, sequences from different samples can be identified in the same run. Multiplexing increase the throughput and efficiency, and cost per data obtained is reduced. The technique has shown its efficacy in analysis of polymorphic DNA. This is used in determining the allelic frequency of any gene in any given population. Though the technology has formed the basis of one of the Next Generation Sequencing platform and there are possibilities of improvement in technique to make it more useful.
Disadvantages of pyrosequencing
Although pyrosequencing is a popular sequencing technique, however the method has some limitations too. It may encounter the problem, when a long stretch of homopolymer is available. Such error may misinterpret as rare operational taxonomic unit (OTU). Several new strategies are employing now to combat these noises. In homopolymeric regions, nonlinear light response is generated followed by incorporation of 5-6 similar dNTPS. This leads to difficulty in determing the actual number of nucleotide incorporated. Excessive research has indicated incorporation of 10 identical adjacent nucleaotide due to apyrase. However specific software algorithms should be utilized to prevent such errors. Short read length is also a limitation of this techniques as some bacteria need full 1.6 kb sequencing of 16S rRNA gene to be reliably identified, however generally near about 500 bp sequencing is considered to be sufficient for distinguishing large number of bacteria.
Conclusion
Pyrosequencing is a newly emerging technique used in Next Generation Sequencing platforms. It can generate about 400 Mb data in a 10 hr run in a single machine. It is a useful technique, currently being used for generating high throughput sequencing data, allelic discrimination; SNP detection and pathogen detection, metagenome study and in future several enhancements in the technique may be useful in increasing its applicability in emerging areas of science.
(The authors are faculty, Department of Genetics, Barkatullah University, Bhopal)
(Courtesy: Current Trends in Biotechnology and Pharmacy, July 2016)