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Research Techniques Made Simple Next-Generation Sequencing: Methodology and Application

Research Techniques Made Simple Next-Generation Sequencing: Methodology and Application. Ayman Grada 1 Kate Weinbrecht 2 1. Department of Dermatology, Boston University School of Medicine 2. Department of Forensic Sciences, Center for Health Sciences, Oklahoma State University.

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Research Techniques Made Simple Next-Generation Sequencing: Methodology and Application

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  1. Research Techniques Made Simple Next-Generation Sequencing: Methodology and Application Ayman Grada1 Kate Weinbrecht2 1. Department of Dermatology,Boston University School of Medicine 2. 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 preparation • Sequencing and imaging • Data analysis • Within each step, the individual platforms have methodological differences that make them unique.

  4. Template Preparation • The DNA (or cDNA) you wish to sequence is fragmented • Adapter fragments are ligated onto the ends of the DNA fragments • The prepared library is clonally amplified • Ion Torrent™: Emulsion PCR • MiSeq™: Cluster generation

  5. Sequencing and Imaging • Sequencing by synthesis • Library fragments act as a template off of which a new DNA fragment is synthesized • Sequencing occurs through a cycle of washing and flooding the DNA fragments with nucleotides in a sequential order • As 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 reads • Alignment to a reference sequence or de novo alignment • Analysis of compiled sequence

  7. General Applications of NGS • The applications of NGS seem almost endless • Resequencing of the human genome to identify genes and regulatory elements involved in disease • Whole-genome sequencing of different species for comparative biology analyses • Sequencing of microbial species to identify novel virulence factors involved in pathogenesis and spread of disease • Gene expression studies using RNA-seq allow researchers and clinicians to visualize expression in sequence form • As NGS continues to grow in popularity, it is inevitable that novel applications will continue to appear

  8. Clinical Applications of NGS • Whole-exome sequencing • The 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 research • Exome sequencing can facilitate the discovery of disease-causing mutations • Targeted sequencing • Sequencing that specifically targets regions of the genome that are of interest to researchers or clinicians • Targeted sequencing is more affordable and yields much higher coverage of genomic regions of interest • Sequencing panels can be developed to target specific genomic regions or disease-causing mutation hotspots

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