Beginner’s guide to next generation sequencing

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 identification, cancer and rare disease research, 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.

NGS workflow

All types of NGS follow a similar workflow. The first step is nucleic acid extraction, either DNA or RNA. This nucleic acid material must be prepared for sequencing by converting it into libraries. Library preparation involves adding adapters, which allow the samples to be indexed (barcoded and identified).

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 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. 

IDT offers a diverse line of predesigned and custom products to support:

• Whole genome sequencing from high-quality and low-quality samples (e.g., FFPE, cfDNA, ssDNA, etc.) from nanogram down to picogram input ranges
• Whole exome sequencing from high-quality and low-quality samples using hybridization capture probes (smaller targeted panels for a subset of loci are also available)
• Targeted DNA sequencing from high-quality and low-quality samples using multiplexed PCR panels
• Metagenomic and metatranscriptomic analysis from complex, highly diverse samples for microbial populations (e.g., soil, gut, skin, etc.)
• Whole transcriptome sequencing from varying quality of RNA samples ranging between picogram to nanogram input ranges
• Targeted RNA sequencing using hybridization capture technology without the need for ribodepletion or poly(A) mRNA selection
• Methylome analysis from single-stranded, bisulfite-converted DNA down to picogram inputs, as well as single-cell methylation sequencing (methyl-seq) applications
• Targeted methyl sequencing using hybridization capture technology
• Chromatin studies for chromatin immunoprecipitation (ChIP) or Hi-C (chromosome conformation capture) technologies
  
  

NGS applications

Whole genome sequencing 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.

IDT offers a variety of predesigned amplicon sequencing panels and hybridization capture panels for targeting oncology genes, the 16S rRNA gene, SARS-CoV-2, among others. They are suitable for germline or somatic variant calling, and some are suitable for analysis of differential gene expression. IDT also offers custom amplicon sequencing panels and custom hybridization panels that are supported by our technical experts who can help guide the design process and answer any questions that may arise. Whether performing target discovery or analyzing a known set of targets, IDT has workflows available for many applications.

Each type of NGS method comes with its own unique benefits and challenges. Contact us to choose the right one for you and your experiment. 

Learn more about NGS

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.

For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. RUO23-1727_001