Determining the order of nucleic acids in biological samples is an integral component of a wide variety of research applications. DNA sequencing has come a long way since the days of two-dimensional chromatography in the 1970s. With the advent of the Sanger chain termination method in 1977, scientists gained the ability to sequence DNA in a reliable and reproducible manner3. Two decades later the short read, massively parallel sequencing technique revolutionized sequencing capabilities and launched the “next generation” in genomic science. Massively parallel sequencing is a stepwise reaction series that consists of (a) a nucleotide addition step, (b) a detection step that determines the identity of the incorporated nucleotides on each fragment being sequenced, and (c) a wash step that may include chemistry to remove fluorescent labels or blocking groups. In essence, next-generation sequencing platforms viz., Illumina, Roche/454 FLX, ABI SOLiD and Ion Torrent conduct sequencing and detection simultaneously rather than as distinct processes. Recently, the 3rd generation sequencing methods viz., SMRT (Single Molecule Real Time) sequencing (PacBio) and Nanopore Sequencing are gaining attention as they are based on sequencing single DNA molecules1.

The major applications of NGS technologies are genome sequencing and resequencing, transcriptomics, exome capturing, study of epigenetic regulation, development of molecular markers and evolutionary studies2. The NGS technologies have been successfully utilized in breeding of several crop plants. Results from whole genome sequencing and assembling process of ML-365 finger millet cultivar yielded 1196 Mb covering approximately 82% of total estimated genome size. Genome analysis showed the presence of 85,243 genes and one half of the genome is repetitive in nature4. Two wheat cultivars contrasting for their salinity stress response were subjected to RNA-Sequencing. Using the SNP polymorphism detected, 157 candidate SNP primer pairs were designed. Further confirmation using polymerase chain reaction (PCR) analysis, 17 SNP markers were developed5.

However, the benefits of developments in the field of NGS tools can only be harvested by integrating the genome, transcriptome techniques and bioinformatics tools. This is going to be a challenging task in coming years. The future holds bright for whole genome sequencing, as the cost of sequencing is expected to decrease further and also the developments in the third-generation sequencing techniques have the potential to decrease the complexity by easily assembling more complex genomes.

References: 

1BHADAURIA, V., 2017, Next-generation sequencing and bioinformatics for plant science, Wymondham: Caister Academic Press. 

2DAVID, V. L. K. AND REPKOVA, J., 2017, Application of Next-Generation Sequencing in plant breeding, Czech J. Genet. Plant Breed., 53: 89-96.

 3HEATHER, J. M. AND CHAIN, B., 2015, The sequence of sequencers: The history of Sequencing DNA, Genomics, 107(1): 1-8. 

4HITTALMANI, S., MAHESH, H. B., SHIRKE, M. D., BIRADAR, H., UDAY, G., ARUNA, Y. R., LOHITHASWA, H. C. AND MOHANRAO, A., 2017, Genome and transcriptome sequence of finger millet (Eleusine coracana (L.) Gaertn.) provides insights into drought tolerance and nutraceutical properties. BMC Genom., 18: 1-16.

 5KIM, S. H., KIM, D. Y, YACOUBI, I. AND SEO, Y. W., 2021, Development of single-nucleotide polymorphism markers of salinity tolerance for Tunisian durum wheat using RNA sequencing, Acta Agric. Scand. B Soil Plant. Sci., 70(1):28-44.