NGS Workflow Steps

NGS workflow steps

Understanding the basic steps in the NGS workflow

Preparing for the NGS workflow

Next-generation sequencing workflows start with nucleic acid isolation, followed by library preparation. Libraries are sequenced on Illumina sequencing systems, designed to support a wide range of applications and throughputs. Data generated are then analyzed to gain insights.

Learn the basics of each step and discover how to plan your NGS workflow.
icon nucleic acid extraction
Step 1: Nucleic acid extraction

The overall workflow for an NGS experiment starts with the isolation of genetic material. Nucleic acids are isolated from samples such as bulk tissue, individual cells, or biofluids. After extraction, most NGS workflows require a QC step. We recommend using UV spectrophotometry for purity assessment and fluorometric methods for nucleic acid quantitation.

icon library prep
Step 2: Library preparation

The library preparation process involves converting a genomic DNA sample (or cDNA sample) into a library of fragments which can then be sequenced on an NGS instrument.

Learn more about library prep

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Step 3: Sequencing

Nucleotides are read on an Illumina sequencer at a read length (length of a DNA fragment that is read on a sequencer) and depth (number of ”reads” that are obtained per sample) that’s recommended for a particular use case.

Illumina uses proven sequencing by synthesis (SBS) chemistry that detects single bases as they are incorporated into growing DNA strands. Several sequencing platforms are available to support a broad range of throughputs and applications, so regardless of your research questions, they can help you get answers faster with simple push-button workflows.

Explore the resources below to learn more:

Intro to SBS technology
Intro to SBS technology

Learn about Illumina next-generation sequencing technology, which allows for massive parallel sequencing.

XLEAP-SBS chemistry
XLEAP-SBS chemistry

The NextSeq 1000 and NextSeq 2000 Systems are powered by XLEAP-SBS chemistry – our fastest, highest quality, and most robust sequencing by synthesis (SBS) chemistry to date.

Benchtop sequencers

Compare benchtop sequencers to see what’s best for your research goals.

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Step 4: Data analysis and interpretation

Bioinformatics tools are used to make sense of the series of As, Ts, Gs and Cs, or reads, that leave an Illumina sequencer.

NGS analysis is now very accessible. Some instruments have built-in ready-to-use onboard data analysis that makes it easy for new users without a bioinformatics background or the budget to hire additional lab staff. The Illumina Connected Software portfolio offers versatile, integrable, and accessible data analysis solutions to drive high-impact research.

Explore the resources below to learn more:

Bioinformatics for beginners
Bioinformatics for beginners

You no longer need to be a bioinformatics expert to analyze your NGS-based experiment data. Learn how innovative features like push-button analysis and automation are making data analysis easy for all researchers.

Learn more about NGS data analysis
Learn more about NGS data analysis

Our sequencing data analysis software helps you spend more time doing research, and less time configuring and running analysis workflows.

Gene expression and regulation bioinformatics eBook
Gene expression and regulation bioinformatics eBook

This e-book outlines the NGS analysis workflow and provides an overview of Illumina Connected Software solutions available for analyzing gene expression and regulation data.

Featured workflow: RNA sequencing

Additional resources

NGS tutorials

With videos, online training, and technical bulletins, explore our NGS tutorials for tips and best practices related to library prep, sequencing, and data analysis.

NGS applications

No matter your research focus, NGS can play an important role in pursuing the answer to a variety of biological questions using a wide array of published methods for diverse sample types.

Have questions about using NGS?

If you have questions about NGS for your specific research focus, we’d love to help. Our specialists can answer any questions and recommend the best solution for your setup.

Speak to a specialist