DNA sequencing with next generation sequencing (NGS): how it works
DNA sequencing determines the order of the bases that make up DNA. It provides information about organisms in areas as diverse as population genetics, epidemiology, organism identification, genotyping, rare variant detection, oncology diagnostics, gene editing confirmation, and gene-environment interactions.
NGS is a high-throughput technology that determines the sequence of a sample all at once by using parallel sequencing. Traditional Sanger sequencing determines the sequence of a sample one section at a time. Sequencing thousands of gene fragments simultaneously with NGS reduces time and cost associated with sequencing and increases the coverage quality and data output.
All types of NGS follow a similar workflow. The first step is to extract genomic material, either DNA or RNA. This genomic material must be prepared for sequencing by converting it into libraries, which facilitate the sequencing process. Library preparation includes adding adapters, which allow the samples to be indexed (barcoded and identified) so that multiple samples can be combined in the same sequencing run (multiplexed). If targeted NGS is being performed, an enrichment step selects for regions of interest. Illumina sequencing is performed using sequencing-by-synthesis on a flow cell. Short-read sequencing generates 100–300 base pair lengths called reads. The reads go through quality control and are aligned to a reference genome before being evaluated for significant characteristics.
Types of NGS
There are many types of NGS; it is most commonly used to evaluate DNA, RNA, DNA-protein interactions, and methylation. Comprehensive data can be gleaned with whole genome sequencing (WGS) or whole transcriptome sequencing (WTS, RNA-seq), or sequencing can be focused with targeted NGS or targeted RNA-seq. The method chosen for a given experiment depends largely on what you hope to learn from the experiment.
NGS has a variety of applications. WGS is used when comparing genotypes and a comprehensive evaluation of the genome is needed, such as when researching rare diseases. Whole exome sequencing is a fast and cost-effective approach to interrogating protein-coding genes in a genome and is often used for tumor normal sequencing. Amplicon sequencing is often used for genotyping by sequencing in agriculture and to validate CRISPR-Cas9 genome edits. ChIP-Seq and Methyl-Seq provide information about the epigenome and gene regulation. Each type of NGS method comes with its own unique benefits and challenges. Talk to a scientific application specialist to choose the right one for you and your experiment.
All of these points are examined in detail with recommendations and rationale provided in the NGS 101 guide. This extensive application guide provides chapters on NGS workflow, as well as types of sequencing and applications. Data analysis and new sequencing platforms are also discussed. The guide is written by IDT scientists, and is free—simply download it here.