DNA sequencing represents one of the most dynamic areas of bioinstrumentation offering an increasing technology diversity rather than coalescence around a single basic approach. Advancement in DNA sequencing has provided the flexibility to perform a wide range of applications, including de novo sequencing, resequencing of whole genomes and target DNA regions, metagenomics, RNA analysis, and ultra-deep amplicon sequencing.

The current generation of DNA sequencing platforms offers various sequencing approaches such as whole genome sequencing, whole exome sequencing, amplicon sequencing to name a few, and the decision on which platform to ultimately go with depends on the needs of the researcher and the data produced, namely transcriptomics, genomics, and epigenomics.

Advances in NGS

The introduction of next-generation sequencing (NGS) technologies has brought about a major transformation in the way scientists extract genetic information from biological systems, revealing limitless insight into the genome, transcriptome, and epigenome. This ability of NGS has catalyzed a number of important breakthroughs in human disease research. As technology has evolved, an increasing number of innovative sample preparation methods and data analysis algorithms have enabled a broad range of scientific applications.

Next-generation sequencing is capable of producing a large amount of sequence data at a reduced cost compared to previous sequencing methods, and the same sequence data can be applied to both copy number variation analysis and other applications such as SNP, indel detection and noninvasive prenatal testing (NIPT), which is used to test for chromosomal abnormalities. Targeted resequencing and whole genome sequencing are the prospective technologies as various targeted disease-based NGS assays and panels are emerging and use of personalized medicine transforms with the understanding of individual's whole genome imprint.

Second Opinion
DNA Sequencing - A Powerful Tool in Genomics

The advent of DNA sequencing has significantly accelerated biological research and discovery.

The sequencing of the human genome along with related organisms has been one of the largest scientific endeavors in the history of mankind. The information garnered from sequencing has provided raw data for the exploding field of bioinformatics, where computer science and biology live in symbiotic harmony.

Impact of NGS in Genomics

The requirement for electrophoretic separation of DNA fragments for reading DNA sequence content in Sanger-based sequencing was the primary bottleneck for throughput with this method, increasing time and limiting the number of reactions that can be run in parallel. Next generation or deep sequencing (NGS) refers to a new, versatile technology of DNA sequencing that permits high-throughput highly adaptable whole genome-scale assays at reasonable cost and high accuracy.


NGS technology finds applications in research as well as diagnostic fields. Whole genome sequencing, investigation of genome diversity, metagenomics, epigenetics, discovery of non-coding RNAs and protein-binding sites, and gene-expression profiling by RNA sequencing are some of the research-based applications. Discovery of new microorganisms and viruses by using metagenomic approaches, analysis of viral genome variability within the host (i.e., quasispecies), and detection of low-abundance antiviral drug-resistance mutations in patients with human immunodeficiency virus (HIV) infection or viral hepatitis are all possible with NGS.

The major clinical diagnostic/IVD applications of NGS are detection of germline variants in cancer as well as other genetically inherited disorders, BRCA1/2 mutation detection in familial breast and ovarian cancers, non-invasive prenatal testing (NIPT), pharmacogenomics and personalized medicine, HLA typing and pre-implantation genetic diagnosis/screening (PGD/PGS). At metropolis, we are offering a variety of sequencing-based assays in the areas of predictive diagnostics (e.g., BRCA1/2), drug resistance detection (e.g., HIV Drug Resistance), genetic disorders (e.g., Beta Thalassemia), pharmacogenetics (Abacavir Hypersentitivty in HIV-1), and companion diagnostics (EGFR, Kras Mutation).

Additional Requisites

Besides NGS instrument and technical expertise, NGS workflow demands strong bioinformatic pipeline, bioinformaticians, experienced geneticist for medical remarks and reporting and genetic counseling services for analyzing and interpretation of sequencing data.

Dr Anurita Pais
Head-Cytogenetic Department,
Metropolis Healthcare Limited<

One noticeable trend is the shift to targeted resequencing, which has overtaken de novo sequencing as the most widely-investigated NGS application. Targeted resequencing involves comparison with a previously generated reference sequence. This allows the variation within the sample sequence to be determined, enabling detection of genetic variants known to play a role in disease, a prerequisite for genomics-based diagnostics. Sequencing vendors are directing their efforts towards improving system performance and extending read lengths and more chemistry kits are available to support target enrichment, an expanding menu of amplicon assays, targeted human DNA sequencing and targeted RNA sequencing. Furthermore, some vendors are developing novel IT platforms to expand the range of analytical tools available to users.

While the latest high-throughput sequencing instruments are capable of massive data output, NGS technology is highly scalable. The same underlying chemistry can be used for lower output volumes for targeted studies or smaller genomes. This scalability gives researchers the flexibility to design studies that best suit the needs of their particular research. Continuous innovations and developments that are aimed at higher throughput, increased accuracy, and affordable costs have made NGS a promising technology. Developments in pre-sequencing, cloud computing, and NGS bioinformatics solutions are major opportunities for development in this segment.

Challenges. With all the advantages that potential NGS brings to research and diagnostics, it also has several pitfalls that need to be addressed. The first problem encountered for diagnostics was the massive amounts of data generated. Large-scale sequencing is on the rise with an increasing number of centers lining up to undertake ambitious projects that demand huge volumes of DNA data. Another challenge is the high cost of acquiring equipment, software and consumables needed for NGS. Analysis and storage of data is another problem faced in the field of data output. The amount of data produced per sequencing cycle on NGS platforms runs into the gigabytes that require specialized high power computers for quick, effective processing and analysis.

Advent of Third-Generation Sequencing Methods

Numerous third-generation sequencing methods are at various stages of commercial development, including electrochemical and semiconductor-based detection systems and laser-excited fluorescence. The aim of most third-generation processes is lower cost and faster overall throughput. Whereas NGS methods feature myriad short reads, third-generation approaches typically emphasize a few very long reads. This simplifies the software challenge of overlapping the oligonucleotide strands to assemble the complete target sequence, reducing the computer time and amount of oversampling.

Nanopore sequencing. This third-generation sequencing technology has two key advantages over second-generation technologies - longer reads and the ability to perform real-time sequence analysis. The single-molecule techniques used by this technology have allowed further study of the interaction between DNA and protein, as well as between protein and protein. Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale. Benefits of nanopore-based sequencing also include detection of epigenetic markers which is not possible using chemical methods as well as cost reduction vis-à-vis traditional methods.

New technologies on the horizon have demonstrated major leaps in innovations in the field of DNA sequencing - the ability to use individual molecules without any library preparation or amplification, the identification of specific nucleotide modifications, and the ability to generate longer sequence reads. The third-generation sequencing methods are proving to be the most versatile way of sampling wet chemistry and biochemistry in real time with unmatched spatial resolution and high (even single-molecule) sensitivity, giving faster overall throughput. These developments will facilitate future research in many fields, make data analysis easier, and further reduce sequencing costs.

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