Impact of Next-Generation Whole-Exome sequencing in Molecular Diagnostics
Next-Generation Sequencing technology provides remarkable opportunities to characterize personal or individual genome sequencing and to identifying mutations relevant for disease diagnosis and therapy. The Whole-Exome sequencing using Next-Generation Sequencing (NGS) is promoting popularity in the human genetics community due to the reasonable costs, feasible data quantity and forthright interpretation of analysis results. This review discusses methods and the challenges in the analysis of NGS data and their potential applications in clinical diagnosis in the Next-Generation Exome sequencing.
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Sequencing
There are many Next Generation Sequencing sequencing platforms available, postdating classical Sanger sequencing methodologies. Other platforms include Roche 454 sequencer and Life Technologies SOLiD systems, the Life Technologies Ion Torrent and Illumina's Illumina Genome Analyzer II (defunct) and subsequent Illumina MiSeq, HiSeq, and NovaSeq series instruments, all of which can be used for massively parallel exome sequencing. These 'short read' NGS systems are particularly well suited to analyse many relatively short stretches of DNA sequence, as found in human exons.
Microarray-based genotyping
Microarrays use hybridization probes to test the prevalence of known DNA sequences, thus they cannot be used to identify unexpected genetic changes.[10] In contrast, the high-throughput sequencing technologies used in exome sequencing directly provide the nucleotide sequences of DNA at the thousands of exonic loci tested.[12] Hence, WES addresses some of the present limitations of hybridization genotyping arrays.
Whole-genome sequencing
Exome sequencing is only able to identify those variants found in the coding region of genes which affect protein function. It is not able to identify the structural and non-coding variants associated with the disease, which can be found using other methods such as whole genome sequencing.There remains 99% of the human genome that is not covered using exome sequencing. Presently, whole genome sequencing is rarely practical in the clinical context due to the high costs and time associated with sequencing full genomes. Exome sequencing allows sequencing of portions of the genome over at least 20 times as many samples compared to whole genome sequencing, at the same cost.For translation of identified rare variants into the clinic, sample size and the ability to interpret the results to provide a clinical diagnosis indicates that with the current knowledge in genetics, exome sequencing may be the most valuable.