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EMT Next Generation Sequencing in Helath and Science
- 1. EMERGING MEDICAL TECHNOLOGIES: NEXT GEN SEQUENCING Cynthia N Perry, PhD Assistant Academic Dean of Admissions Assistant Professor, Molecular Medicine Department of Medical Education Foster School of Medicine
- 3. HUMAN GENOME • 3 billion bases arrayed in a unique order, • with ~20,000 genes that direct the synthesis of all the proteins • ~1/1,000,000th of the Human Genome • Polymorphisms or SNPs which differ between people • May influence traits • Possible medical importance
- 4. HISTORY OF DNA SEQUENCING • 1st Generation = Sanger Sequencing –2 reads (forward & reverse) • 2nd Generation = Next Generation Sequencing –Millions of reads • 3rd Generation = Single Molecule Sequencing
- 7. FROM SANGER TO NEXT-GEN SEQUENCING
- 10. APPLICATIONS IN MEDICINE- INFECTIOUS DIESEASE Clinical Test for TB Patient • Take a sputum sample • Begin culture • ID drug susceptibility ID by NGS
- 11. APPLICATIONS IN MEDICINE - CANCER
- 12. APPLICATIONS IN MEDICINE - PRENATAL CARE
- 13. BROAD APPLICATIONS • Paternity testing • Wildlife conservation • Herbal supplement validation • Ancestral migration patterns • Origins of traditional medicine
- 14. PORTABLE SEQUENCER (MINION) • Pocket-sized, portable device for biological analysis • Up to 512 nanopore channels • Simple 10-min sample prep available • Real-time analysis for rapid, efficient workflows • Adaptable to direct DNA or RNA sequencing
- 16. Next Generation Sequencing In the palm of your hand Practical module Miki Wang, Cynthia Perry, and Jorge Cervantes
Editor's Notes
- No disclosures or conflicts of interest
- Comprehensive genomic profiling assays provide extensive coverage of variants and immunotherapy biomarkers and increasingly enable physicians to provide cancer patients with more informed treatments. Now that these assays are commercially available for the first time, laboratories have the option of running them in house, which can positively impact quality and crucial turnaround time. https://youtu.be/KiQgrK3tge8
- The Human Genome Project was a 13-year-long, publicly funded project initiated in 1990 with the objective of determining the DNA sequence of the human genome within 15 years. In its early days, the Human Genome Project was met with skepticism by many people, including scientists and nonscientists alike. One prominent question was whether the huge cost of the project would outweigh the potential benefits. Today, however, the overwhelming success of the Human Genome Project is readily apparent. Not only did the completion of this project usher in a new era in medicine, but it also led to significant advances in the types of technology used to sequence DNA. One particularly striking finding of the Human Genome Project research is that the human nucleotide sequence is nearly identical (99.9%) between any two individuals. However, a single nucleotide change in a single gene can be responsible for causing human disease. Because of this, our knowledge of the human genome sequence has also contributed immensely to our understanding of the molecular mechanisms underlying a multitude of human diseases. Furthermore, a merging of cytogenetic approaches with the human genome sequence will continue to propel our understanding of human disease to an entirely new level.
- Broadly speaking, there are two types of DNA sequencing: shotgun and high-throughput. Shotgun (Sanger) sequencing is the more traditional approach, designed for sequencing entire chromosomes or long DNA strands with more than 1000 base pairs. It involves a rapidly expanding firing pattern to read the DNA in short fragments of 100 to 1000 base pairs, which are then overlapped with a computed analysis system. Second generation sequencing or Next Generation Sequencing came along in 2007 and is a High-throughput method and has led to the rapid acceleration of DNA sequencing and broadened knowledge in the field. It is able to produce thousands of sequences simultaneously, which lowers the cost and increased the speed of the technique significantly. in some ways, next gen sequencing or NGS has been superseded by a new generation of sequencing that looks at single molecules.
- This method uses DNA polymerase, the same enzyme used in DNA replication, to produce DNA sequence information. DNA polymerase binds to a single-stranded DNA template and adds DNA bases to the 3′ end of the complementary DNA strand it synthesizes. When DNA polymerase randomly incorporates a fluorescently labeled ddNTP base, synthesis terminates. This step produces a mixture of newly synthesized DNA strands that differ in length by a single nucleotide. Each strand is labeled at the 3′ end with a fluorescently labeled dideoxynucleotide base. The DNA mixture of newly synthesized DNA strands that differ in length by a single nucleotide is separated by electrophoresis. The electropherogram results show peaks representing the color and signal intensity of each DNA band. From these data, the sequence of the newly synthesized DNA strand is determined,
- Development of NGS technology has fundamentally changed the kinds of questions scientists can ask and answer. Innovative sample preparation and data analysis options enable a broad range of applications. For example, NGS allows researchers to: Rapidly sequence whole genomes Zoom in to deeply sequence target regions Utilize RNA sequencing (RNA-Seq) to discover novel RNA variants and splice sites, or quantify mRNAs for gene expression analysis Analyze epigenetic factors such as genome-wide DNA methylation and DNA-protein interactions Sequence cancer samples to study rare somatic variants, tumor subclones, and more Study microbial diversity in humans or in the environment The capacity to sequence all 3.2 billion bases of the human genome has increased exponentially since the 1990s. In 2005,1.3 human genomes could be sequenced annually. Nearly 10 years later, the number has climbed to 18,000 human genomes a year. Today an individual’s genone can be synthesized in about a day.
- In principle, the concept behind NGS technology is similar to CE sequencing. DNA polymerase catalyzes the incorporation of fluorescently labeled deoxyribonucleotide triphosphates (dNTPs) into a DNA template strand during sequential cycles of DNA synthesis. During each cycle, at the point of incorporation, the nucleotides are identified by fluorophore excitation. The critical difference is that, instead of sequencing a single DNA fragment, NGS extends this process across millions of fragments in a massively parallel fashion. It delivers high accuracy, a high yield of error-free reads Library Preparation—The sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5′and 3′adapter ligation. Alternatively, “tagmentation” combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process. Adapter-ligated fragments are then PCR amplified and gel purified. Cluster Generation—For cluster generation, the library is loaded into a flow cell where fragments are captured on a lawn of surface-bound oligos complementary to the library adapters. Each fragment is then amplified into distinct, clonal clusters through bridge amplification. When cluster generation is complete, the templates are ready for sequencing. Sequencing—Illumina technology uses proprietary reversible terminator–based method that detects single bases as they are incorporated into DNA template strands. As all four reversible terminator–bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias and greatly reduces raw error rates compared to other technologies.6,7 The result is highly accurate base-by-base sequencing that virtually eliminates sequence context–specific errors, even within repetitive sequence regions and homopolymers. Data Analysis—During data analysis and alignment, the newly identified sequence reads are aligned to a reference genome. Following alignment, many variations of analysis are possible, such as single nucleotide polymorphism (SNP) or insertion-deletion identification, phylogenetic or metagenomic analysis, and more.
- Understanding the sequences of DNA can be applied in various settings. It now forms the base of biologic research and is applied in biotechnology, forensic biology, virology and medical diagnoses. Researchers are already able to use the results of DNA sequencing to compare long lengths of DNA. In some cases, this may include looking at segments of over a million bases to compare differences in the sequencing. This information can reveal important information about the role of certain DNA patterns and susceptibility to health condition or response to medical treatment. The routine use of DNA sequencing as a diagnostic tool for the general practitioner remains a possibility for the future, but there are some ways that sequencing is already being used for medical purposes. For example, DNA sequencing is currently used for cancer patients to help identify the type of cancer that is present, which directs the treatment decisions for the patient. Similar methods are currently in development for other health conditions that are likely to have a genetic element, such as cardiovascular disease and diabetes.
- Fetal DNA found in mother’s bloodstream can be isolated and tested for inherited mutations, birth and developmental defects, and even gender
- Antibiotic resistance can be identified and the correct drug therapies implemented earlier By sequencing the genomes of tusks seized from elephant poachers, this work identified the location of the poaching “hotspots” and may allow enforcement officials to concentrate their efforts in those locations. Thus, sequencing technology is helping to protect elephants from poachers and stem the illegal trade of ivory worldwide Many people are turning to herbal supplements, seeking a more natural way to stay healthy. Although widely consumed, data from next generation sequencing has questioned whether some herbal supplements actually contained the ingredients on the label. Researchers sequenced the contents of 44 herbal supplements. Only two of the products tested contained 100% authentic ingredients. There are many theories as to how people populated the North and South of America. NGS data has suggested an interesting model. The mitochondrial genome was determined from 2 infants found at an ancient burial site in Alaska. Interestingly, the mtDNA was more closely similar to mitochondrial DNA from a 500 year-old Incan child mummy found in Argentina. This has huge implications as to how humans populated the Americas Ayurvedic medicine is based on a whole-body holistic approach. SNP analysis of genetic markers was able to accurately categorize a population of Indian males into the three groups used in Prakriti. One form of Ayurveda, focusing on the interplay between lifestyle and environment and how this may influence disease. This suggests that the ancient practice of Ayurveda may have a genetic basis and scientifically validates this ancient medicine.
- 1.8nm biological pores ssDNA is threaded through pore and individual current traces are detected by self contained voltage detector Higher error rates 10-15% Loooong reads up to 2mb Can seq RNA