Abstract: Next Generation Sequencing (NGS) techniques retain the potential to open up avenue to identify novel therapeutic target guided drug to pursue and proving as an efficient alternative technology for denovo drug design. Its remarkable applications are established in relation to therapeutic diagnosis and treatment of multifactorial diseases including cancer. The NGS accurately predicts cellular reactions at genomic, transcriptomic or proteomic level. Its impact in novel anticancer drug designing relies on its therapeutic approaches, multiplexing of samples and high diagnostic sensitivity for genetic and epigenetic biomarkers. Development of new therapies and drug usually takes longer time and require recruiting considerable pools of patient. NGS cut down cost of drug development and time by using its unseen potential to identified specific therapeutic target and new pathophysiological pathways involvement in cancer, helped in designing of targeted drug and correct evaluation of its deleterious side effect. Furthermore, improved gene and disease interaction validations techniques changed entire cancer therapy methods. This is a critical review of multiple approaches of NGS in development of anti-cancer drug including biomarkers based diagnosis and recent trends of targeted capture technology based personalised medicine in cancer therapy. Additionally, emerging paradigm in disease diagnosis based on state-of-art third generation sequencing technologies have also discussed.
INTRODUCTION
Next generation sequencing emerged as a promising approach for therapeutic diagnosis and treatment of complex pathophysiological conditions including cancer. At global level, cancer is considered as major health problem and now become leading cause of mortality. Cancer is a complex disease process accompanied by large numbers of correlated genetic alternations affecting deregulation of cell cycle1. According to World Health Organization (WHO) 8.8 million deaths were caused by cancer in 2015 along with a prediction of 12 million cancer death per year by 20302. Breast, colorectal, lung and cervix are most frequently observed cancer in women whereas lung, prostate, colorectal, stomach and liver are main cancer types causing death in men2. In both developing as well as developed countries, lung and breast cancer are primary causes of deaths in males and females respectively3-5. Furthermore, liver and stomach cancer in males and cervical cancer in females were also significantly reduce survival rate of cancer patients as reported by National Cancer Institute-NIH6.
In view of recent scenario in therapeutics, Next Generation Sequencing (NGS) technologies are recognised as a high-throughput sequencing and data analysis approach, essentially needed for therapeutic diagnosis and treatment of cancer. The NGS allows assessment of tumour genomic at individual gene or transcript level using massive parallel run and multiplexing of samples7,8. The longstanding challenges in clinical diagnosis and treatment of malignancies represent variability of treatment and gradual development of resistance to medication at sub-clones level9. The ability of NGS to work with comprehensive landscape from identification of genetic alternations, the main cause of multiple resistances makes it the right approach for designing the right drugs for cancer for right patients, with specific dose, at defined time interval.
History of sequencing methods: Advent of First Generation Sequencing (FGS) technology was started with the development of chemical method by Maxam-Gilbert10 and dideoxy method by Sanger11. In the Maxam and Gilbert method, terminally labelled DNA fragments were cleaved chemically at adenine, guanine, cytosine or thymine and reaction products were separated by gel electrophoresis based on size of fragments to determine nucleotide sequence12. However, dideoxy method of Sanger determined DNA sequence through base specific termination of DNA synthesis using chain termination of 2’,3’-dideoxy and arabinonucleoside analogs11,12. Sanger method of DNA sequencing was adopted as primary sequencing technology among FGS for clinical as well as research13. The FGS was primarily based on radioactive or fluorescent materials and due to limited sequencing potential, gradual changes in applications of sequencing technologies in clinical medicines lead to dramatic change in sequencing scenario resulting in the development of Second Generation Sequencing (SGS) technology now referred as Next Generation Sequencing (NGS) technology13.
NGS: A new vision for therapeutics: The significance of next generation sequencing technologies lies in the comparative analysis of clinical samples in a much faster and inexpensive way. The NGS methods are based on several techniques including, (1) Micro-chip based electrophoretic sequencing, (2) Sequencing by hybridization, (3) Real-time sequencing and (4) Cyclic-array sequencing14. The working mechanism is composed of certain basic steps that include genomic template preparation for downstream sequence analysis, generation of short sequence reads, alignment of reads on reference sequence, assembly of sequence from aligned reads15. Most NGS technologies utilize sequencing by synthetic16 approaches and have been used by various commercial platforms that include Roche/454 life science, Illumina/Solexa, Applied Biosystem/SOLiD, Polonator and Helicos Biosciences and single molecule sequencing as tabulated in Table 117,18.
The NGS approach for designing targeted small-molecule cancer drugs capture the sense of excitement along with reducing the existing designing burden on researchers in cancer treatment. There have been many existing problems and challenges associated with traditional designing of drugs. These challenges represented as complexity of anti-cancer drug discovery process, low precision level of target identification, high cost of drug synthesis and clinical trials, limited knowledge of underlined molecular mechanism and lack of validated biomarkers for characterisation of tumour type19. Furthermore, the process of drug design and development (Fig. 1) has high level of failure rate at the stage of clinical trials. Another big challenge is to understand the way to overcome drug resistance that results relapse of tumour growth and considered as major hurdle in cancer therapy20. Significant progress has been marked by the development of anti-cancer drugs such as imatinib and erlotinib and leukemia patients who are not responding the imatinib, sequencing of patient genome profile used to recommend before changing the therapy that can be done by SGS technologies21.
The NGS has wide range of applicability in the field of cancer research, specifically since cancers are mainly caused by gene mutations.
| Fig. 1: | Drug discovery process based on next generation sequencing |
| Table 1: | Comparative overview of different NGS platforms |
The NGS technology enables researcher for the identification of gene mutations, characterisation of tumour type, diagnosis of tumour progression by biomarker prediction. Finding of Ley et al.22 reported whole-genome sequencing study on acute myeloid leukemic cells22. The impact of FLT3 gene mutation has been identified in acute myloid leukemic patients and solid tumour exomes of breast and colorectal cancer were first identified using NGS technology23-25. Multiple mutated genes were associated with malignancies depending on its location and type. In myeloma, BRAF, NRAS and KIT were observed as mutation causing genes. Such genes have targeted for reducing metastatic growth26. Four subtypes of breast cancer and their mutated genes have been discriminated based on exome analysis. The frequencies of TP53 and HER2 mutations were found to be highest in tumour27. Using NGS, breast cancer progression has been estimated by finding difference in read length of CAG repeats in terms of intra-tumour genetic heterogeneity of androgen receptor gene. It has been observed that shorter length of CAG repeats may have protective role against breast cancer28. The discovery of biomarkers like BRCA1, BRCA2, HER2, PR and ER are known to be extremely significant in molecular profiling, tumourigenicity and targeted drug designing29. The NGS plays potential role in finding mutation in heterogeneous population of cancer cells through biomarker detection30.
Based on the analysis of genetic mutations, NGS technologies facilitate discovery of precision medicine in oncology. Broadly, there are three ways that confer NGS utility in cancer therapy. First, diagnosis of tumour type determined by mutations leads to genetic alternations. Second approach predicts targeted gene therapy against specific tumour type. Third strategy finds mutations that cause resistance to targeted therapy31. The NGS technologies integrate genomics, transcriptomic and epigenomic mutations in cancer biology as well as classify various types of cancers for early diagnosis and targeted therapy32.
DEVELOPMENT OF NGS TECHNOLOGIES IN RELATION TO CANCER
NGS and pharmacogenomics: The NGS has wide spectra for drug development in connection to pharmacogenomics, deals with the study of association between genetic variation and drug response for disease treatment33. It correlates drug efficacy and toxicity with genomic variation in drug targets that contributes in improving treatment response. In cancer, genetic variations of patient must be examined by considering both acquired (somatic) and germline (inherited) mutations due to their significance in drug efficacy. Analysis of somatic mutations plays peculiar role in enhancing treatment efficacy by defining genetic alterations during tumour development resulted in prediction of potential drug target such as mutations in TP53 and CYP19 are used to predict genetic constituent of breast cancer34. On the other hand, germline mutations find pharmacokinetic property of drug to understand treatment response to targeted therapy35. In oncology, there have been mammoth of challenging tasks, which increases the need of advanced NGS technologies and use of statistical analysis methods. The analysis of large amount of data generated from multiple samples of cancer patients and identification of rare genetic variants from those samples are major challenges in pharmacogenomics. The NGS technologies provide fast and robust approach to tackle such challenges36. Moreover, NGS determines molecular pathways associated with metastasis, finds polymorphisms in genes causing multidrug resistance and targets potential drugs against specific genetic mutation. Integration of genome-wide association study (GWAS) and NGS have great significance on survival rate of cancer patients with the advancement in cancer therapy37.
NGS methods in cancer therapeutics: Early diagnosis of cancer development is now possible with discoveries of advanced NGS technologies. This leads to easier cancer genomic profiling and provides targeted therapy. For genomic profiling of cancer patients, formalin fixation and paraffin embedding are two commonly used pathological biopsy media38. The sensitivity and accuracy of profiling cancer genome is influenced by steps of genomic data generation protocol that includes pre-analytical methods (data collection, storage, extraction and manipulation), library preparation, sequencing and variant calling. Variations have observed in preparation issues of pre-analytical methods based on type of sample, selection of sequencing-based assays as illustrated in Table 239.
To understand small genetic alterations in cancer patients, whole genome sequencing, whole exome sequencing, targeted RNA panel, transcriptomie sequencing are used30. Whole Genome Sequencing (WGS) detects copy number variants with high resolution, regulatory regions like promoters and enhancers and determines intergenic regions40. This approach allows researchers to examine cancer genome for identification and categorization of novel mutations41.
| Table 2: | Common preclinical and sequencing assays in cancer genome profiling |
However, Whole Exome Sequencing (WES) is focused to cover 1-2% of entire genome42,43. The coverage of WES is up to 95% of exons much higher than WGS. The WES detects both somatic and germline mutations in cancer patients43. Transcriptome sequencing of cancer genome profiling has been carried out using mRNA expression analysis. In addition to gene expression, the analysis of DNA alterations makes the transcriptomic analysis method more effective44. The transcriptome sequencing has immense significance for non-coding RNA (i.e., miRNA, siRNA, piRNA and IncRNA) detection based biomarker development44. Transcriptome sequencing method assesses epigenome, proteome and metabolome in a broader way44. Targeted panel sequencing associated with precision oncology in which gene abnormalities have been identified in the panel of 20-500 genes build on amplicon based or hybridisation based techniques45. It has been used in detection of Single Nucleotide Variants (SNVs) and small insertions/deletions (Indels operation) for cancer therapeutics46. Targeted panel sequencing is quite useful in providing high depth and high exon coverage which are two critical factors for consistent variant calling. The depth of coverage defines repetition of a specific base in sequencing and alignment to a reference genome45,46. Apart from that, exon coverage also depicts spanning by atleast one sequencing read in terms of percentage47,48. The formalin-fixed paraffin-embedded tissue has been used in targeted panel sequencing method. Different aspects of NGS at epigenomic, transcriptomic and genomic level are depicted in Fig. 2 while comprehensive view of whole genome sequencing, whole exome sequencing, transcriptome analysis and targeted panel sequencing is shown in Table 3.
Other methods of NGS are de-novo sequencing, non-coding RNA sequencing and epigenomics sequencing. De-novo sequencing deals with alignment of reads to generate sequencing of complete genome specifically when reference genome is unavailable49. In non-coding RNA sequencing, the regulation of differential gene expressions has been analysed as silencers or repressors50. Epigenomic approach includes methylation sequencing, ChIP sequencing and ribosome profiling51,52.
| Fig. 2: | Different approaches of Next generation sequencing technologies |
| Table 3: | Comparative aspects of common NGS sequencing techniques |
| CNV: Copy number variation, SNVs: Single nucleotide variants | |
The study of cytosine methylation profile in the region of hetrochromatin and promoters, suggests deeper insight into the regulation of gene expression patterns53. Epigenomic sequencing allows identification of methylation up to single nucleotide level and DNA fragmentation up to 100-150 bp followed by construction of standard libraries for NGS analysis53. Chromatin immune-precipitation (ChIP) sequencing enables diagnosis of any diseased state through the study of protein-DNA or protein-RNA interaction54. Ribosome profiling mainly focus on active mRNA fragments captured by ribosome during translation processes provides overall activity of cell at specific time scale and facilitates identification of active forms of proteins that modulate cellular processes55.
APPROACHES USED BY NGS TECHNOLOGIES IN TUMOUR IDENTIFICATION
From the past two decades, next generation sequencing technologies emerged as a high-throughput technology to analyze biological information relatively at very low cost and provided novel platform for biological research. The NGS allowing the researchers to perform almost any type of analysis and identified potential therapeutic target at genomics, transcriptomics or proteomics level by considering genetic or epigenetic factors associate with disease.
The urgent need of sequencing for understanding holistic nature of complex disease leads to the foundation of Human Genome Project (HGP) primarily based on Sanger method of sequencing. However, extremely high cost of genome sequencing was remained as a major barrier to further implement it in the area of clinical and personalised medicines56,57. The introduction of next generation sequencing technology in the 2000s dramatically drop down the cost of genome sequencing by almost 50000 folds, as it was roughly $300 million estimated for generating the first initial draft of human genome sequence under Human Genome Project58. Figure 3 clearly shown fortunes of NGS techniques and downfall of cost over year. At present, one can get complete sequence of cell after expending approximately at $1000 or below59.
| Fig. 3: | Comparative fortune of NGS techniques and downfall of cost |
In last one decade, the next generation sequencing has undergone several technological up-gradation and has evolved as a reliable platform for the current era of genomics. The first automated genome sequencing machine (AB370) was launched by Applied Biosystem in 198760. AB370 was able to detect 96 bases simultaneously with 500k bases in a day considering maximum read length capacity of 600 bases as compared with the current AB3730 machine that can detect up to 2.88M bases per day61.
Basic workflow of NGS technologies: Therapeutic applications recruited NGS guided approaches mostly based on sample type and diagnostic questions to be addressed for a specific disease in defined pathophysiological condition. NGS approaches can be grouped into ‘DNA-seq’, ‘RNA-seq’, ‘ChIP-seq’ and ‘methyl-seq’ analysis based on the sample type62,63. However, the diagnostic approach and overall treatment protocols may vary among different techniques. The present review highlights the base methodology (Fig. 4) of sequencing in reference to one of the most reliable Illumina MiSeq and Ion Torrent PGM machine64.
Step 1: Sample/library preparation: Genomic materials isolated from diseased tissue fragmented either enzymatically or mechanically up to required fragment size, inputted to sequencer. However, sample amplification may be preferred for 4-10 cycles using PCR in most of the cases and based on the initial amount of genomic material65. The genomic information produced in form of ‘reads’, stored using .SRA, .FASTA or .FASTAQ file formats.
Step 2: Quality check: Performed to remove any bad quality reads, with quality score less than standard cut-off defined by scale of Phred quality score66. Phred quality score (Q-score) used to measure the base calling accuracy of sequencers and indicates the incorporation of incorrect bases. Q score, Qscore is measured as negative log of base calling error probabilities, Pbcp (Eq. 1)67:
Qscore = -10 log10×Pbcp |
(1) |
Equation 1 explained probability of incorrect base insertion 1 in 1000 run of sequencers. Phred quality score for base is 30 represents 99.9% base accuracy66. Table 4 list base calling error probabilities, Pbcp with Phred score, Qscore.
Step 3: Mapping/assembly of reads: This is one of the crucial steps of NGS technologies marked by either finding of overlapping zone among all reads of DNA-seq or RNA-seq data followed by construction of contigs or mapping of reads against known reference genome. Overall performance has been measured by calculating length (maximum, average, combined) and N5068. N50 best represented as set of largest contigs whose sum of length is more than 50% length of the assembly69.
| Table 4: | Quality check score for sequencer: Base calling error probabilities, with Phred score,Qscore |
| Fig. 4: | Underline steps of next generation sequencing technology with associated software |
However, some of the researchers mentioned that assembly accuracy is difficult to measure69.
Step 4: Downstream analysis: Overall flavour of NGS technologies are to find out pattern of novel gene expression from cell type in particular therapeutic condition. DNA-seq mostly used to find out homozygous and heterozygous single nucleotide polymorphism (SNPs) and mutations, locus of insertion and deletion (InDels), structural variants, and, copy number variations (CNVs)59,70,71. While, RNA-seq used for finding splicing variant regions, differential expression of genes, gene regulatory networks, signaling pathways and networks70,72. Furthermore, several standard platforms developed for downstream analysis includes SAMtools73, GATK74 and cummeRbund75 which have been specially designed for cuffdiff output for RNA-seq data.
In cancer studies, most of tumours are resulted due to disturbance of genetic state of cell. This disturbance may be of genetic or epigenetic in nature. The variance between tumour and normal cell lines well demonstrated with high accuracy and sensitivity by using GATK, SAMtools, mpileup and Isaac variant caller76.
NGS AND TARGET CAPTURE TECHNOLOGY
Next generation sequencing technology utilized several approaches for parallel sequencing of high-throughput data but most of them contribute in ‘Research Use Only’ (RUO)77. Application of NGS in therapeutic diagnosis needs consistent accuracy and high performance as per the guidelines of state agencies regulating the clinical laboratory78. Several potential biomarkers have been identified at genomic or transcriptomic level by researchers for clinical diagnosis of cancer (Table 5) and further validated through molecular diagnosis, based on the applications of NGS technologies.
Traditional methods of sequencing required considerable amount of DNA or RNA for clinical identification of therapeutic target. However, NGS technologies need comparatively small amount of genomic material for screening of several genes simultaneously70. Sequencing of known list of biomarkers for clinical screening provides alternative and efficient approach for routine analysis of samples of therapeutic importance. Several commercial gene panels or kits are available, designed to screen list of genes of particular therapeutic interest. The content of NGS panel is an important consideration for screening list of genes to be targeted and sequenced79.
Target Capture Technologies (TCT) has been designed to take advantage of known biomarkers and target for small potential set of therapeutic genes80. The TCT mostly depends on two factors, first is sample type and second is quality and quantity of DNA or RNA81. Table 6 represents the list of important target capture technologies, underlying principle and amount of nucleic acid obligatory for analysis.
NGS: DRUG DESIGNING TO CANCER THERAPY
In the last five decades, the approvals of new drugs by competing authorities have almost remains constant while the cost of clinical diagnosis to treatment of diseases, has increased to three times measuring in the yearly scale of 199082.
| Table 5: | Globally identified selected biomarkers for tumour as per the guidelines of ‘The American Society of Clinical Oncology’ (ASCO) |
| Source: https://www.cancer.gov/, NIH: National Cancer Institute | |
| Table 6: | Target capture technologies, area of enrichment application and amount of nucleic acid required for analysis |
| *Depending on the version of kit-TruSeq Amplicon (recent version v1.5), illumina, North America | |
| Table 7: | List of approved drugs in relation to cancer therapy |
| Source: http://www.fda.gov | |
The major cause of this bottleneck can be grouped into technological and administrative barriers. Technological barrier represented as fall back of developmental efficiency of new drug and limitation of production models while administrative barriers have been adversely affective the overall process including strict regulatory and experimental framework, continuously rising cost of scientific query and complexities of patent process and its effect after expirations83.
To overcome all barriers in the process of drug development and designing of new models for cancer therapy, both biotechnology and pharmaceutical based interdisciplinary companies, have started utilizing the concept of comparatively more efficient NGS guided genome sequencing based approaches to overcome the technological and administrative bottlenecks84. Researchers has been utilizing the concept of sequencing based approach and remarkably several new discovered drugs have been approved for further clinical diagnosis and therapeutic uses84. Recently, more than 100 drugs of pharmaceutical importance in distant therapeutic area have been listed by US Food and Drug Administration (FDA) (http://www.fda.gov). Table 7 show the advancement in cancer therapy and development of anticancer drugs.
NGS guided approach has been provided promising platform for pharmaceutical industries but still the success rate to launched new drug has remained constant and surprisingly, it has been observed that only one out of ten drugs qualified the preclinical testing criteria85, 86.
| Fig. 5: | Number of clinical trials registered for cancer according to Food and Drug Administration Amendments Act-2007 |
| Source: https://clinicaltrials.gov/ct2/results/details?term=cancer | |
In the year of 2010, Ginsburg et al. noted that 45 different drugs failed in phase III of clinical diagnosis and testing stage, causing the loss of huge investment against average recorded cost of about $100 million per drug in phase III87.
CHANGING PARADIGM: NGS GUIDED PERSONALISED ONCOGENIC TREATMENT
Over last two decades, there has been many fold increase in application of NGS technologies in diagnosis of potential genes alterations, splicing sites and epigenetic guided alternations considering multifactorial nature of disease88. These technologies are primarily used to identify the causing factors of a specific therapeutic condition that leads to the discovery of new diagnostic techniques for designing of novel drugs. Total 60046 hits were observed (Fig. 5) against searching keyword ‘cancer’ over the database of ClinicalTrials which was launched to implement ‘Food and Drug Administration Amendments Act’ of 2007 (FDAAA) clearly highlights the use of NGS in research. Large number of comprehensive cancer projects have been launched globally such as The Cancer Genome Atlas (TCGA)89 (https://cancergenome.nih.gov/), apply genome analysis techniques for diagnosis of molecular basis of cancer along with comprehensive identification of co-related changes of cancer sub-types88, International Cancer Genome Consortium (ICGC)90 (http://icgc.org/) working on 50 different type of cancer of clinical and social importance and Cancer Genome Project (CGP)91 (http://www.sanger.ac.uk/genetics/CGP/) involves in diagnosis of novel set of genes in cancer development.
Researchers have discovered plethora of diagnostic techniques for diagnosis of novel and rare mutations associated with a particular therapeutic condition. The NGS has been successfully recruited in most of the diagnostic purposes, including common cancer like lung cancer, prostate cancer and breast cancer48.
To date, number of oncogenic biomarkers (Table 5) have been identified and characterised in application of clinical practices as per the clinical practice guideline issued by ‘The American Society of Clinical Oncology’ (ASCO)92. Current interest of researchers hasmoved in the area of personalised diagnosis and treatment of cancer. Next generation sequencing revolutionized an era of genome sequencing and medical science provided, comparatively much faster tools for screening of biomarkers, sequencing of whole genome or targeted gene of interest, reliable identification of genetic interaction, which were unidentified with traditional cytogenetic techniques74.
Recent studies have clearly witnessed the efficient transformation of therapeutic workflows of NGS technologies into comparatively very high accuracy using WGS, WES or Epigenetic based analysis with broader clinical impact in personalised medicine. Walsh et al.93 demonstrated the clinical diagnostic capability of NGS to target germ line mutations from patient suffering from primary level of carcinoma93. In other experiment, Holbrook et al. (year) identified the genes signature (AURKA, EGFR, FGFR2, KRAS, NET and PIK3CA) in gastric cancer based on Illumina sequencing technology and successfully used this technique on personalised treatment94. Now it is possible to have comprehensive disease history of patient for personalised treatment of therapeutic regimens based on sequencing of normal and tumour genomes. However, in spite of tremendous utility in cancer therapeutics, NGS based technologies have number of challenges that need to be addressed for further clinical investigation and its implementation for personalised medicines and treatment of cancer.
CHALLENGES OF NGS AND FUTURE TECHNOLOGY FOR MALIGNANCY
Next generation technologies revolutionised diagnosis processes of complex diseases including cancer and established Omics approach for understanding of disease complexities and further taking holistic approach of treatment. Cancer research based on NGS has witnessed exceptionally fast, more reliable approaches within the accepted guidelines of clinical as well as molecular diagnosis of malignancies. Recent achievements of NGS platform can be best represented in diagnosis and identified therapeutic targets for breast cancer95-97, ovarian cancer98, lung cancer99, melanoma100, colorectal cancer97 and head and neck cancer101. Although, NGS personified plethora of information for diagnosis and treatment of malignancy, still there have been many computational and clinical challenges remains that need to be addressed.
Computational barrier can be best described in the use of statistical approaches to identify set of differentially expressed genes, estimate overall accuracy of mapping and measure alignment of reads, derived from especially repetitive segments of DNA. Current algorithms of NGS technologies are difficult to explain for differential identification of genes, new isoforms or rare splicing junctions102. One of our research works in connection to identify potential set of probes guided subset of genes considering high throughput screening of microarray replicates for breast cancer explained the advantages and disadvantage of statistical approaches103. The work was considered three methods, fisher discriminate ratio104, vector norm105 and t-test (paired)106 used to revalidate the potential set of gene. Furthermore, there is a need of high performance computational architecture to stores, analyses and interprets the NGS data107. Additionally, Clinical challenges are mainly concerned with design of novel anti-cancer drugs and applications of personalised medicines utilising concept of targeted therapy. However, sometimes biomarkers based therapy selection process made to be very difficult, if tumour spreading resulted into sub-clones and every sub-clone identified by separate genetic biomarkers108.
The NGS based outcome are generally difficult to explain particularly in connection to cancer research. To overcome the heterogeneous complexity of inter-regulated tumour biomarkers, Third Generation Sequencing (TGS) technologies appears to lead the entire clinical diagnosis and treatment processes. The most significant aspect of TGS lies in the capability of interpreting even a single altered nucleotide from small genomic sequence instead of working with considerable amount of patients data65. Nanopore guided sequencing techniques emerged as promising candidate among all TGS techniques and its first version (MinION sequencer) has already been distributed among selected group of researchers for diagnostic and testing purposes in 2014109. Single Molecule Real Time (SMRT) Sequencing is one of the most recognised TGS techniques introduced in 2009 and its main advantage includes production of long-reads over the other sequencing techniques110. Researchers have been trying to address this problem by introducing the concept of ‘Hybrid technologies’ by combining short and long-read sequencing technology that may work as milestone in cancer research110. Single Molecule DNA Sequencing (SMDS) and Direct RNA Sequencing (DRS), is the first commercial available third generation sequencers allows up to 50 parallel run with capacity of almost 30×106 reads in each run111,112. Third generation sequencing technology hitherto catalysed the genome assemblies and sequencing speed many folds, still there have been many issues that need improvement particularly in specificity of statistical approaches, accuracy in output error rate and efficiency of result interpretation .
CONCLUSION
The advancement in sequencing technology has exceedingly supported the therapeutic diagnosis and treatment of human diseases. Omic approach of Next Generation Sequencing (NGS) provides much faster, cost effective and comparatively more realistic way to decode life mysteries and improve life qualities. Precise analysis of genomic data has opened fascinating opportunities in medical sciences including designing of novel drugs, identification of therapeutic targets, use of targeted capture technologies to speed up overall disease screening processes and reliable use of personalised medicine. Interesting to mention that, NGS shifted the bottleneck of diagnosis process from sequencing of large-reads of genomic or transcriptomic sequence to computational management of clinical data.
Applications of NGS to deal with inter-tumour and intra-tumour heterogeneity turned up into an idea that each tumour is unique and showing distinct mutation profiles even derived within a single tumour. Additionally, clinical method to identify malignancies (biopsy) only covers about 55% of tumour mutation profiles. Therefore, treatment of disease was atomistic in nature. NGS technology has provided more robust and sensitive platform followed by diagnosis of specific tumour type and taking holistic approach of treatment.
In the last decade, pathway based analysis of genetic variations in human cancers has been centre of attraction. Now-a-days, NGS based applications are integrated with Genome-Wide Association Study (GWAS) to detect any systematic changes and co-regulated heterogeneity in genome expression profile of tumour. Apart from addressing the issues of tumour heterogeneity and pathway based analysis, target based treatment has been continuously emerging as reported by several clinical trials. It is now, believed that rapid expanding of NGS based clinical trials and identified biomarkers leading to further advancement in drug efficacy.
In recent years, technological advancements of next generation sequencing have revolutionized whole medical sciences and clinical approaches dealing with human cancers. Short read-alignment processes have been shifting towards large-read mapping and subsequently effective library preparation using SMRT and Nanopore based third generation sequencing techniques have further improved the disease classification and early diagnosis processes. It is hoped that in the next decade, the muti-targeted and ultra deep sequencing techniques shall become more effective and way of diagnosis process and treatments will be improved further specifically with respect to cancers.
SIGNIFICANCE STATEMENT
Next Generation Sequencing (NGS) technologies providing a new platform for efficient identification of cancer biomarkers guided therapeutic targets, improved and reliable approaches for designing of novel anticancer drugs and time effective application of cancer therapy. Several NGS based omics approaches have been recruited for successful identification of cancer biomarkers and designing of personalized medicines even if, patients stop responding in conventional therapy. The NGS technologies have number of potential significances over traditional approach including remarkable changes from morphological to genetical identification of tumor type, targeted based drug designing and comparatively real and holistic view of overall treatment process.
ACKNOWLEDGMENTS
All the authors greatly acknowledge the logistic support of their respective Universities and Institutes.