10. Clinical Application of HT-NGS High-Throughput Next Second Generation Sequencing (HT-NGS) has transformed genomic research by decreasing the cost of sequencing and increasing the throughput. Now, the focus is on using NGS technology for diagnostics and therapeutics. A number of recently launched NGS systems open new opportunities for disease research, including infectious disease studies, detection of rare variants, understanding the genomic complexity of cancer, and conducting epigenetics studies. NGS promises to facilitate genome-wide human population structural variation studies by uncovering all of the common and rare genetic variation in human populations. Indeed, the “1000 Genomes Project” has made great progress to date toward this goal. With a comprehensive genetic map of all human variation produced by HT-NGS, researchers will be able to perform more detailed experiments to detect genetic variation underlying the response to medicines. HT-NGS platforms have also found application in high throughput mutation detection and carrier screening using a method called functional genomics fingerprinting (FGF). This approach uses a selective enrichment of functional genomic regions (the exome, promoterome, or exon splice enhancers) in response to the discovery of causal mutations for disease and drug response. Target enrichment based on microarray has also allowed the parallel, large-scale analysis of complete genomic regions for multiple genes of a disease pathway, and for multiple samples simultaneously, thus providing an efficient tool for comprehensive diagnostic screening of mutations. Carrier screening by HT-NGS is also feasible in the general population with severe recessive childhood disorders and in mutation detection associated with autosomal-recessive cerebellar ataxia, by combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval.
10.1 Genetic Mutations in Mendelian Disorders Mendelian disorders are genetic diseases that show Mendelian pattern of inheritance. Traditionally, karyotyping and fluorescent in-situ hybridization (FISH) was used to identify gross genetic abnormalities while in recent years microarrays have provided better resolution. Since most of the genetic abnormalities in a number of Mendelian disorders are small (bp-kbp), HT-NGS with a single nucleotide resolution would be better suited for identifying them. Moreover, HT-NGS systems could be used to sequence the entire exome or targeted exonic regions along with the associated 5’, 3’ and intronic regions for focused genetic variation detection. Additionally, multiplexing capabilities of HT-NGS technology would allow for sequencing small regions of multiple samples in a single lane/ well. Exome sequencing has been used to identify genes underlying rare Mendelian disorders; Miller syndrome [QUERY Reference format – Author name] and Kabuki syndrome.
10.2 Epigenetics The HT-NGS technologies offer the potential to substantially accelerate epigenomic research (the study of heritable gene regulation that does not involve the DNA sequence itself but its modifications and higher-order structures), including posttranslational modifications of histones, the interaction between transcription factors and their direct targets, nucleosome positioning on a genome-wide scale and the characterization of DNA methylation patterns. Histone modification and methylation of DNA are two important epigenetic mechanisms that regulate the transcriptional status of genes. Using ChIP-Seq (chromatin immunoprecipitation and direct sequencing) technology, posttranslational modifications of histones and the location of transcription factors can be studied at the wholegenome level, whereas methylated DNA immunoprecipitation (meDIP) and bisulphite protocols can be used to study the methylation of DNA itself. For example, using ChIP-seq on HT-NGS platform, the binding sites for a transcription factor (TF) and the human growth-associated binding protein (GABP alpha) were directly sequenced instead of being hybridized on a chip-array thus unraveling the wide and intricate gene pathways regulated by PPARG (Peroxisome proliferator-activated receptor gamma) gene and predicting the de novo motif discovery. The ChIP-seq approach was recently used to identify binding sites of two transcription factors, STAT1 (Signal Transducers and Activators of Transcription 1) and NRSF (Neuron-Restrictive Silencer Factor) in human cells. Both studies compared their findings with those generated by ChIPchip, demonstrating that ChIP-seq had better resolution and required fewer replicates.