1. Research Techniques Made Simple: Next-Generation Sequencing:
Methodology and Application
Ayman Grada1
Kate Weinbrecht2
1. Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USA
2. Department of Forensic Sciences, Center for Health Sciences, Oklahoma State University, Tulsa, Oklahoma, USA

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