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.
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.
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.
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
Learn about Illumina next-generation sequencing technology, which allows for massive parallel sequencing.
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.
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
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
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
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.
Step 2 in NGS Workflow: Sequencing
During the sequencing step of the NGS workflow, libraries are loaded onto a flow cell and placed on the sequencer. The clusters of DNA fragments are amplified in a process called cluster generation, resulting in millions of copies of single-stranded DNA. On most Illumina sequencing instruments, clustering occurs automatically.
In a process called sequencing by synthesis (SBS), chemically modified nucleotides bind to the DNA template strand through natural complementarity. Each nucleotide contains a fluorescent tag and a reversible terminator that blocks incorporation of the next base. The fluorescent signal indicates which nucleotide has been added, and the terminator is cleaved so the next base can bind.
After reading the forward DNA strand, the reads are washed away, and the process repeats for the reverse strand. This method is called paired-end sequencing.
Step 3 in NGS Workflow: Data Analysis
After sequencing, the instrument software identifies nucleotides (a process called base calling) and the predicted accuracy of those base calls. During data analysis, you can import your sequencing data into a standard analysis tool or set up your own pipeline.
Today, you can use intuitive data analysis apps to analyze NGS data without bioinformatics training or additional lab staff. These tools provide sequence alignment, variant calling, data visualization, or interpretation.
Get started with your workflow
Design an NGS workflow that’s right for you, process your samples, and generate your first NGS data set.
Access resource and tools
NGS Workflow Finder
Take the guesswork out of your next workflow. The NGS Workflow Finder provides personalized solution recommendations and resources so you can sequence with confidence.
Find your NGS workflow todayFeatured workflow: RNA sequencing
RNA isolation
There are several 3rd-party options for RNA isolation kits depending on your sample type.
Library preparation
Sequencing
MiSeq System
For smaller panels: targeted resequencing, metagenomics, small genome sequencing, targeted gene expression profiling, and more.
NextSeq 1000 & 2000 Systems
For larger panels: Targeted sequencing, RNA-Seq, single-cell methods, exome sequencing, and more.
Illumina Technology Course
This course provides and overview on preparing libraries, cluster generation, sequencing workflow essentials, and data analysis. Begin your journey here and see how Illumina sequencing technology can accelerate research discoveries.
Start CourseAdditional 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.