AssignmentApplied Genomics and Proteomics

MTECH (BIOTECHNOLOGY) 3rd SEM 2014-2016

Topic: Applications of NGS in Research

SUBMITTED TO: - SUBMITTED BY:-Dr. Vipin Singh RENU RAWATDr. Chanderdeep Tandon Enroll: A0510714028Dr. Archana Chaturvedi Roll No.: 1027 Amity Institute of Biotechnology

INTRODUCTIONDNA sequencing is the process by which we extract the sequence of A, C, T, and G of the entire DNA. Since the introduction of the Sanger sequencing method in the 1970s [1], DNA sequencing technology has enabled enormous advances in molecular biology and genetics. This technology has been used for many projects, such as the Human Genome Project, Rice Genome Project and Swine Genome Project, as well as genome projects of many other species. However, there were a few disadvantages of this sequencing technology. These include its low throughput, high cost and operation difficulties. Due to these disadvantages Sanger sequencing have limited its use in deeper and more complex genome analyses[2]. The recent introduction of next-generation sequencing (NGS) technology, with its high-throughput capacity and low cost, has largely overcome these problems, and these technologies have been applied in various fields of life sciences, including forensics, disease diagnosis, agrigenomicsand ancient DNA analysis. In this article, the use of NGS technology in forensic science is reviewed with the aim of providing a reference for future frontier research and application in forensic science.

APPLICATIONS1. IN CANCER RESEARCH [3] With the development and improvement of new sequencing technology, next generation sequencing (NGS) has been applied increasingly in cancer genomics research over the past decades. NGS is used to identify novel and rare cancer mutations, detect familial cancer mutation carriers, and provide molecular rationale for appropriate targeted therapy.Identification of novel cancer mutations using NGSNGS technologies have enabled efficient and accurate detection of novel and rare somatic mutations. NGS has been successfully employed to identify novel mutations in a variety of cancers, including bladder cancer, renal cell carcinoma, small cell lung cancer, prostate cancer, acute myelogenous leukemia, and chronic lymphocytic leukemia. Whole genome sequencing with NGS was used in patients with a rare form of acute promyelocytic leukemia and successfully identified a novel PML-RARA genetic recombination that was undetectable with standard cytogenetic techniques. Gui et al sequenced the exomes of 9 transitional cell carcinoma tumors to find somatic mutations, then screened in tumor samples from 88 individuals with transitional cell carcinoma at different stages and grades. They found 55 notable mutations related to transitional cell carcinoma, 49 of which were first found in bladder cancer. Furthermore, they sequenced the whole exomes of 10 clear cell renal cell carcinomas and screened thousands of genes in an additional 88 samples, ultimately discovering 12 new mutated genes. Both studies were published in Nature Genetics in the same year.In another study, Keller et al. used specific target selection and NGS to identify novel SNPs in genes already associated with glioblastoma. Over 6000 SNPs, including more than 1300 SNPs located in targeted genes, were identified.By using NGS, numerous novel genetic aberrations and associated potential therapeutic targets have been found in many cancers. Many of these studies on new genetic aberrations were summarized by Tran et al.

NGS in hereditary cancer syndrome genetic testingAbout 5% 10% of cancers is hereditary. Genetic testing has been used for hereditary cancer patients for more than ten years in the US and Europe. The development of NGS provided many opportunities for genetic testing. Walsh et al. used target region capture and NGS to detect 21 genes associated with hereditary breast and ovarian cancer. This combined method allowed detection of several kinds of variations, including single nucleotide substitutions (SNPs), small insertions and deletions, and large genomic duplications and deletions. NGS provides a good solution for detecting rare variations. Because it allows testing of multiple genes at once, NGS greatly improves the variation detection rate. Many patients with hereditary cancer have tested negative for genetic variations, but with NGS, it is easier to find causative mutations. In a study of 300 high-risk breast cancer families, Walsh et al. found previously undetected mutations in 52 probands. Ozcelik et al. introduced a method that used long-range PCR plus NGS to detect BRCA1 and BRCA2 and demonstrated that it was useful for BRCA testing. A similar method has also been reported previously by Hernan et al. and De Leeneer et al. NGS for personalized cancer treatmentNGS is also used to improve rationally designed individualized medicine. For example, NGS has been used in the treatment of pancreatic cancer. It has been also used in the detection of epidermal growth factor receptor (EGFR) deletions in non-small cell lung cancer, which showed important pathogenetic and clinical implications for patients with nonsmall cell lung cancer. In addition, it has been used in the detection of PML-RARA fusion gene in acute promyelocytic leukemia, which led to a change of a patients therapeutic schedule. In an inspiring study, genetics researchers at Washington University did whole genome and transcriptome sequencing for a researcher in their team, who had adult acute lymphoblastic leukemia. The cancer relapsed twice in 10 years from the time of first diagnosis. Then, his colleagues found a clue about the disease through RNA sequencing. Their results showed that a normal gene, FLT3, was wildly active in the leukemia cells. Luckily, however, the drug sunitinib, which is approved to treat advanced renal cancer, inhibits FLT3. With sunitinib treatment, his blood counts appeared more normal. This is a very successful case of translating NGS into clinical practice.

Table1: Number of cancer genomes sequenced [4]

Detection of circulating cancer DNA by NGSRare mutations in circulating DNA have long been used to detect somatic mutation for cancer diagnosis and management. It is difficult to identify rare mutations in tumor suppressor genes like TP53, which is highly mutated throughout the gene. NGS proved to be the cost-effective method to detect and measure the allele frequency of TP53 and other tumor gene mutations in the plasma. Forshew et al. developed a tagged amplicon deep sequencing (TAmSeq) method that used NGS and designed primers to amplify approximately 6000 bases that covered the selected regions of cancer related genes, including EGFR, TP53 and KRAS. By using plasma samples, they showed that the method could identify mutations in TP53 at allelic frequencies of 2% to 65%, thereby demonstrating that it is feasible to sequence large regions of circulating DNA by NGS. Thus, continuous dedication to apply NGS in clinical oncology practice will enable us to be one step closer to personalized medicine.

2. AGING RESEARCH [5]NGS can help unravel the biological and genetic mechanisms of aging, longevity and age-related diseases. Using NGS genes contributing to aging and age-related diseases are being determined. Eg: Transcription factors of the FoxO family play a conserved role in controlling longevity downstream of the insulin pathway. FoxO orthologs are already known to extend life span in invertebrates and SNPs in the FoxO3gene are associated with extreme longevity in humans. Genome-wide analysis of FoxO3 binding sites in neural stem cells using next-generation sequencing technologies identified a network of FoxO3 targets involved in stemness and aging. This is possible by following ways:a. Genome sequencing and resequencing: Genome resequencing is a powerful approach to identify genetic variants associated with longevity and/or age-related diseases. Already a number of studies have examined the association between gene variants and human longevity. For example, two recent studies in different populations reported gene variants in FOXO3Aassociated with human longevity [6].GWAS of longevity and age-related diseases using resequencing techniques will play a pivotal role to identify new alleles that determine longevity, susceptibility to age-related diseases and how environmental factors interact with genes to influence these phenotypes.b. Transcriptional profiling: It has helped to identify biomarkers of aging. NGS platforms allows researchers to characterize the aging transcriptome with exceptional resolution and identify transcripts associated with age as well as with life-extension due to genetic or environmental interventions in order to provide new insights about aging and its underlying molecular and genetic mechanisms. c. DNA-protein interactions (ChIP-Seq): Several studies have highlighted the importance of using ChIP-Chip, by itself or in combination with other approaches, in aging research. One of these discoveries was the finding that histones are modified at the telomeres in senescent human cells. d. Sequencing the epigenome: Epigenetics represents chemical alterations of the DNA and histones that impact on function. Such alterations have been suggested to play an important role in aging. Whole-genome analysis of epigenetic marks at the finer resolution delivered by NGS platforms promise to be of great value to biogerontologists. Epigenetic signatures might also be useful to identify biomarkers of aging and age-related diseases, potentially leading to improved diagnosis and risk prediction of the latter.

Fig: Employing NGS platforms to study age-related changes. During the course of an organisms lifetime, a number of genomic changes occur. NGS allows these changes to be quantified at a whole-genome level. Changes to be DNA, from single nucleotide mutations to large chromosome rearrangements, can be detected (A). Likewise, genome-wide epigenetic changes across the lifespan (or between different lifestyles or diets) can be assayed. Lastly, transcriptional changes with age can be quantified with unprecedented accuracy using NGS (C). Mouse and human figures were drawn using fonts by Alan Carr.

3. DRUG DISCOVERYUtilizing NGS, early target identification can be hastened, new genetic lesions associated with disease can be found, and overall development times associated with therapeutics and diagnostics to newly identified targets can be significantly shortened.Broad applications of NGS to drug discovery [7]:1.Mutation detection: personalized medicine2.ChIP-Seq: target identification and/or validation and compound profiling for epigenetics3.CNV: target identification, personalized medicine, for example, cancer4.Exome sequencing: target identification and/or drug resistance studies, biomarker discovery5.RNA-Seq: target identification and/or validation by studying differential gene or miRNA expression between normal and diseased tissue6.HITS-CLIP: study of RNAprotein interactions7.Ribosome profiling: target identification by measuring protein translation rates using sequencing to identifying ribosomal footprints.8.Small RNA sequencing (e.g. miRNA): biomarker discovery9.Bacterial genome sequencing: target identification, validation and diagnostics to identify new strains and mechanisms of drug resistance.

4. ANTIBODY ENGINEERING [8].Antibody display libraries derived from human PBMCs or hybridomas immortalized from B cell populations have been successfully used in recent decades to isolate binders against a wide range of targets, despite a lack of detailed knowledge of the repertoires (34, 35). With the advent of NGS, analysis of the natural nave repertoires from which libraries have been constructed has become possible. NGS can reveal the sequence space occupied by antibodies in their recognition of antigens.

5. ENVIRONMENTAL DNA RESEARCH [9].The analysis of environmental DNA through the use of specific gene markers such as species-specific DNA barcodes has been a key application of NGS technologies in ecological and environmental research. NGS technologies have facilitated analysis of environmentally derived samples from a variety of ecosystems including freshwater, marine, soil, terrestrial and gut microbiota. The majority of these studies seek to answer the question of what is present in a given environment. Through the use of NGS platforms, researchers have been able to observe the slight changes in community structure that may occur following anthropogenic or natural environmental fluctuations. Several studies have analyzed soil bacterial diversity by examining 16S rDNA amplicons. Results suggest that agricultural management of soil may significantly influence the diversity of bacteria and archaea. Other studies have focused on soil fungal diversity in both forest and agricultural settings by analyzing ITS amplicons. Marine environments have also been the subject of ecological research employing NGS technology. Analyses of marine bacterial communities have been conducted using 18S rDNA and 16S rDNA amplicons. Frias-Lopez et al studied microbial community gene expression in ocean surface waters through transcriptomic sequencing analysis of cDNA libraries. Mou et al. investigated functional assemblages within seawater through a NGS analysis of functional metabolic gene regions within bacterioplankton. Marine eukaryotic microbiota was investigated through NGS analysis of 18S rDNA amplicons.

6. Forensic research [10].NGS technology has become an important analytical tool for many forensic researchers. It is being applied to simultaneously analyzing multiple loci of forensic interest in different genetic contexts, such as autosomes, mitochondrial and sex chromosomes. Furthermore, NGS technology has potential applications in many other aspects of research. These include DNA database construction, ancestry and phenotypic inference, monozygotic twin studies, body fluid and species identification, and forensic animal, plant and microbiological analyses.

Fig: Forensic analysis by next-generation sequencingNGS will potentially influence many aspects of forensic science, including short tandem repeats (STRs) and microRNA analysis, monozygotic twin and mixed stain recognition, Y chromosome and mitochondrial whole-genome studies, forensic microbiological analysis, multiple species identification, and ancestry and phenotype inference. More importantly, high-throughput screening techniques have generated large amounts of data, facilitating a systematic understanding of relationships between molecular components. Therefore, comprehensive genome-wide analysis, in combination with the techniques of genomics, proteomics, transcriptomics and epigenomics, will provide new insights in the field of applied forensics.Through applying NGS technology, multiple results can be obtained simultaneously from biological evidence samples collected from crime scenes, such as STRs, single nucleotide polymorphisms (SNPs) of autosomes, sex chromosomes and mitochondrial genomes, as well as epigenetic information. By integrating all the information, the evidence samples can be used not only for suspect identification but also for inferring the criminal suspects physical, psychological and geographical characteristics, as well as the source population.

Fig: Diverse range of information can be obtained by NGS of biological evidence samples collected from crime scenes

7. IN FUNCTIONAL GENOMICS RESEARCH [11].1. Genome annotation and gene expression profiling: The next generation sequencing-based SAGE method, termed DeepSAGE, greatly simplifies the sample preparation procedure by removing the cloning step and replacing it with emulsion PCR-based amplification; the sequencing is conducted by the 454 protocol that allows multiple samples to be sequenced in a single run at a high depth. Nielsen et al. applied the DeepSAGE protocol to the analysis of the transcriptome of the potato and showed that it was efficient at detecting rare transcripts. 2. Small ncRNA discovery and profiling: ncRNAs are RNA molecules that are not translated into a protein product. This class of RNAs includes transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear and small nucleolar RNA, and microRNA and small interfering RNA (miRNA and siRNA). Recent research has implicated microRNAs, approximately 21-nucleotide-long RNA molecules, as crucial posttranscriptional regulators of gene expression in both animals and plants. High-throughput sequencing of small RNAs provides great potential for the identification of novel small RNAs as well as profiling of known and novel small RNA genes. 3. Protein coding gene annotation using transcriptome sequence data: Next-generation sequencing technologies have the potential for providing much deeper coverage of EST libraries. A recent study used laser capture microdissection to isolate transcripts from the shoot apical meristem of Z. mays followed by cDNA library construction and 454 sequencing of ESTs. The study used a cis-alignment method to annotate more than 25,000 genomic sequences from maize and detect transcription from 400 orphan genes, most of which had not been detected using other approaches.4. Detection of aberrant transcription: Large-scale transcriptome sequencing studies provide a novel means for detecting genome rearrangements in the transcribed portion of the genome. An elegant gene identification signature analysis using paired-end ditag transcriptome sequencing methodology has been developed for the detection of gene fusions and other aberrant transcripts in cancers. The approach involves generation of 18-nt-long tags from both ends of a transcript, which are then concatenated and sequenced by the 454 technology. This strategy is particularly useful for detecting fusion events in cancers, as well as actively transcribed Pseudogenes that are readily distinguishable from their source genomic loci.5. Analysis of epigenetic modifications of histones and DNA: The NGS technologies offer the potential to accelerate epigenomic research substantially. To date, these technologies have been applied in several epigenomic areas, including the characterization of DNA methylation patterns, posttranslational modifications of histones, and nucleosome positioning on a genome-wide scale.6. Study of DNA accessibility and chromatin structure: NGS technologies have been applied to mapping out the positions of nucleosomes and other determinants of DNA accessibility

8. AGRICULTURAL RESEARCH [12].NGS technologies have several potential applications in crop genetics and breeding, including the generation of genomic resources, marker development and QTL mapping, wide crosses and alien gene introgression, expression analysis, association genetics and population biology, as shown here. For instance, sequencing of genomic DNA including bacterial artificial chromosomes (BACs), reduced representation of genome (RRG) or cDNA from the reference genotypes using NGS technologies can provide genomic resources such as ESTs, gene space and genome assembly.These resources have a direct impact on understanding the genome architecture for crop genetics. Another application of NGS is in parental genotyping of mapping populations or of wild relatives, which can accelerate the development of molecular markers, e.g. simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. These markers can be used to construct genetic maps, to identify QTLs and to monitor alien genome introgression in the case of wide crosses. These QTL-associated markers for a trait of interest can then be used in selecting progenies carrying favorable alleles via marker-assisted selection (MAS). To develop the functional or perfect gene-based marker, NGS of cDNAs of contrasting genotypes for the trait of interest can be used to identify candidate genes involved in or associated with the trait. The expression mapping of these candidate genes, together with phenotyping of the segregating populations developed from the contrasting genotypes, will provide expression QTLs (eQTLs) and markers associated with these eQTLs should thus serve as the perfect markers for MAS in crop breeding. Another important application of NGS is in association genetics or population biology, where either genomes or pools of PCR products of thousands of candidate genes can be sequenced in hundreds of individuals using barcodes. The sequence data obtained could then be used to identify SNPs or haplotypes across genes or genomes for use in association genetics and/or population biology.

Fig: Overview of NGS applications in crop genetics and breeding

Table: Applications of NGS technologies in plant genetics and breeding

REFERENCES: 1. Sanger F., Nicklen S., Coulson A.R. DNA sequencing with chain-terminating inhibitors.Proc Natl Acad Sci U S A.1977;74:546354672. Fullwood M.J., Wei C.L., Liu E.T., Ruan Y. Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses.Genome Res.2009; 19:521532.3. Guan YF, Li GR, Wang RJ, et al. Application of next-generation sequencing in clinical oncology to advance personalized treatment of cancer. Chin J Cancer. 2012; 31(10):463-470.4. Upadhyay P, Dwivedi R, Dutt A. Applications of next-generation sequencing in cancer. Curr Sci. 2014:795-802.5. Joao Pedro de Magalhaes, Caleb E. Finch, and Georges Janssens. Next-generation sequencing in aging research: emerging applications, problems, pitfalls and possible solutions. Ageing Res Rev. 2010 July; 9(3): 315323.6. Flachsbart F, Caliebe A, Kleindorp R, Blanche H, von Eller-Eberstein H, Nikolaus S, Schreiber S, Nebel A. Association of FOXO3A variation with human longevity confirmed in German centenarians. Proc Natl Acad Sci U S A 2009;106:270027057. Peter M. Woollard, Nalini A.L. Mehta, Jessica J. Vamathevan, Stephanie Van Horn, Bhushan K. Bonde and David J. Dow. The application of next-generation sequencing technologies to drug discovery and development. Drug Discovery Today Vol 16, Numbers 11/12. June 2011.8. Pascale Mathonet and Christopher G. Ullman, The application of next generation sequencing to the understanding of antibody repertoires. Frontiers in Immunology, September 2013, Vol 4, Article 265.9. Shadi Shokralla, Jennifer L. Spall, Joel F. Gibson And Mehrdad Hajibabaei, Next-generation sequencing technologies for environmental DNA research, Molecular Ecology (2012) 21, 17941805.10. Yaran Yang, Bingbing Xie, Jiangwei Yan, Application of Next-generation Sequencing Technology in Forensic Science, Genomics Proteomics Bioinformatics 12 (2014) 190197.11. Olena Morozova, Marco A. Marra. Applications of next-generation sequencing technologies in functional genomics, Genomics 92 (2008) 25526412. Varshney, R. K., Nayak S. N., May G. D., and. Jackson S. A., 2009, Nextgeneration sequencing and their implications for crop genetics and breeding. Trends Biotechnology 27, 522530.