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Research Techniques Made Simple Next-Generation Sequencing: Methodology and Application Ayman Grada 1 Kate Weinbrecht 2 1. Department of Dermatology, Boston.

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Presentation on theme: "Research Techniques Made Simple Next-Generation Sequencing: Methodology and Application Ayman Grada 1 Kate Weinbrecht 2 1. Department of Dermatology, Boston."— Presentation transcript:

1 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

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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