Presentation on theme: "Next-Generation Sequencing: Methodology and Application"— Presentation transcript:
1 Next-Generation Sequencing: Methodology and Application Research Techniques Made SimpleNext-Generation Sequencing:Methodology and ApplicationAyman Grada1Kate Weinbrecht21. Department of Dermatology, Boston University School of Medicine2. Department of Forensic Sciences, Center for Health Sciences, Oklahoma State University
2 Next-Generation Sequencing Sequencing is a method for determining the exact order of nucleotides in a given DNA or RNA sequence.Next-generation sequencing (NGS) platforms perform massively parallel sequencing, during which millions of DNA fragments are sequenced in unison.Rapid (sequence an entire genome in less than one day)Low cost in comparison to traditional techniques (Sanger sequencing)NGS platforms include:Ion Torrent PGM™ (LifeTechnologies, Carlsbad, CA)MiSeq™ (Illumina, San Diego, CA)Several others not discussed
3 Overview of NGS Methodology The Ion Torrent PGM™ and the MiSeq™ have a common base methodology:Template preparationSequencing and imagingData analysisWithin each step, the individual platforms have methodological differences that make them unique.
4 Template PreparationThe DNA (or cDNA) you wish to sequence is fragmentedAdapter fragments are ligated onto the ends of the DNA fragmentsThe prepared library is clonally amplifiedIon Torrent™: Emulsion PCRMiSeq™: Cluster generation
5 Sequencing and Imaging Sequencing by synthesisLibrary fragments act as a template off of which a new DNA fragment is synthesizedSequencing occurs through a cycle of washing and flooding the DNA fragments with nucleotides in a sequential orderAs nucleotides incorporate into a growing strand of DNA they are digitally recorded as sequence
6 Data Analysis Raw sequence data must undergo several analysis steps Preprocessing to remove adapter sequences and low- quality readsAlignment to a reference sequence or de novo alignmentAnalysis of compiled sequence
7 General Applications of NGS The applications of NGS seem almost endlessResequencing of the human genome to identify genes and regulatory elements involved in diseaseWhole-genome sequencing of different species for comparative biology analysesSequencing of microbial species to identify novel virulence factors involved in pathogenesis and spread of diseaseGene expression studies using RNA-seq allow researchers and clinicians to visualize expression in sequence formAs NGS continues to grow in popularity, it is inevitable that novel applications will continue to appear
8 Clinical Applications of NGS Whole-exome sequencingThe exome consists of only the protein-coding regions of the genome (a little over 1% of the genome)Sequencing of the exome is used in gene discovery researchExome sequencing can facilitate the discovery of disease-causing mutationsTargeted sequencingSequencing that specifically targets regions of the genome that are of interest to researchers or cliniciansTargeted sequencing is more affordable and yields much higher coverage of genomic regions of interestSequencing panels can be developed to target specific genomic regions or disease-causing mutation hotspots
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