Question

The completion of the human genome project in 2003 took 13 years and was mainly achieved using the sanger DNA sequencing technique.Discuss how the Next generation sequencing technique of "sequencing by synthesis"(sometimes called Massive-parallel sequencing ) has radically soeeded up the process of genome sequencing and comment on its clinical application

Answer 1

Next generation sequencing technique of "sequencing by synthesis"

Next-generation sequencing (NGS) sometimes called massively parallel or deep sequencing sequencing technology has revolutionized the biological sciences. The entire human genome can be sequenced within a single day using NGS. NGS enables researchers to perform a wide variety of applications and study biological systems with its ultra-high throughput, scalability, speed, innovative sample preparation and data analysis. NGS platforms perform sequencing of millions of small fragments of DNA in parallel. It is also used to sequence entire genomes or constrained to specific areas of interest, eg. a whole exome or  small numbers of individual genes.

Some adavantages of NGS are

• NGS makes large-scale whole-genome sequencing (WGS)
• Whole exome and transcriptome sequencing using NGS is beneficial in uncovering mutations and pathways associated with rare genetic disease.
• NGS-based RNA sequencing (RNA-Seq) is a powerful method to discover novel RNA variants and splice sites and quantify mRNAs for gene expression analysis.
• Using NGA we can analyze epigenetic factors such as genome-wide DNA methylation and DNA-protein interactions.
• To study cancer samples for  rare somatic variants, tumor subclones, and more
• RNA-Seq, ChIP-Seq and exome sequencing for cancer gene expression analysis and biomarker discovery in clinical research.
• Study microbial diversity in humans or in the environment.

Key sequencing methods in NGS

DNA Sequencing - We can focus or analyze regions of interest with whole-exome, the entire genome and also to study the DNA-protein interactions.

RNA Sequencing - We can sequence the targeted RNA to single-cell and whole transcriptome sequencing.

Methylation Sequencing - Genome wide analysis provide insight into methylation patterns at a single nucleotide level.

Potential uses of NGS in clinical practice

Clinical genetics

There are numerous opportunities to use NGS in clinical practice to improve patient care.

NGS captures a broader spectrum of mutations than Sanger sequencing

The mutations in a human genome comprises substitutions, insertions and deletions of DNA, large genomic deletions of exons or whole genes and rearrangements such as inversions and translocations. Sanger sequencing cannot detect some mutations such as substitutions and small insertions and deletions. Some submicroscopic chromosomal copy number changes such as microdeletions also cannot detect. But these data can derive directly from NGS sequencing data and harvest the full spectrum of genomic variation in a single experiment.

Genomes can be interrogated without bias

NGS is used to interrogate full genomes or exomes to discover entirely novel mutations and disease causing genes. In paediatrics, this could be exploited to unravel the genetic basis of unexplained syndromes.

The increased sensitivity of NGS allows detection of mosaic mutations

NGS sequencing provides a sensitive read-out and used to identify mutations including mosaic mutation which are acquired as a postfertilisation event .

Microbiology

NGS replaced the conventional characterisation of pathogens by morphology, staining properties and metabolic criteria in microbiology with a genomic definition of pathogens.

Oncology

Targeted NGS identifies diffuse glioma genetic markers, supporting use in tumor characterization for clinical research.