AAT Bioquest

What are the 4 steps of next generation sequencing (NGS)?

Posted February 16, 2022


The 4 steps of next generation sequencing (NGS) include nucleic acid isolation, library preparation, clonal amplification and sequencing, and data analysis.

Step 1- Nucleic Acid Extraction and Isolation

Nucleic acid extraction and isolation is a vital first step in next generation sequencing. This is regardless of whether you are sequencing total RNA, genomic DNA, or various RNA types. The extraction method that’s used will depend on the starting material. It is crucial to choose an extraction protocol that’s optimized to yield the maximum amount and highest purity of nucleic acid from the respective sample type. The yield, quality and integrity of isolated nucleic acids are critical for successful sequencing and must be assessed before proceeding to the next step.

Step 2- Library Preparation

Library preparation involves preparing DNA or RNA samples so they can be processed and read by sequencers. This is done by fragmenting the samples to yield a pool of appropriately sized targets, then adding specialized adapters at both ends, which will later interact with the NGS platform. These prepared, ready-to-sequence samples are called “libraries”. A library represents a collection of molecules that can be sequenced. The exact library preparation procedure may differ depending on the reagents and methods used. Regardless of the procedure used, the final prepared NGS libraries must contain DNA fragments of desired lengths with adapters at both ends.

Step 3- Clonal Amplification and Sequencing

Clonal amplification involves amplifying DNA fragments to be sequenced by binding to ion surfaces, beads or flow cells. This helps develop strong fluorescent signals that can be detected by the sequencers. 

Sequencing by synthesis (SBS) is the next step after clonal amplification. In this step the library is loaded onto the sequencer, which then ‘reads’ or detects the nucleotides one by one. 

Step 4 -Data Analysis Using Bioinformatics

This final step involves three stages – processing, analysis, and interpretation of the raw sequencing data generated. A variety of bioinformatics tools are used to process, analyze, and interpret the raw sequencing data and convert it into meaningful information. The exact tools used as well as how the data is processed and analyzed depends on the applications and goals of the NGS assay.

Additional resources

A Next-Generation Sequencing Primer—How Does It Work and What Can It Do?