NGS Workflow Steps

Main Steps in Next-Generation Sequencing

The next-generation sequencing workflow contains three basic steps: library preparation, sequencing, and data analysis. Learn the basics of each step and discover how to plan your NGS workflow.

View an Example Workflow
example NGS workflow infographic

Preparing for the NGS Workflow

Before starting the next-generation sequencing workflow, isolate and purify your nucleic acid. Some DNA extraction methods can introduce inhibitors, which can negatively affect the enzymatic reactions that occur in the NGS workflow. For best results, use an extraction protocol optimized for your sample type. For RNA sequencing experiments, convert RNA to cDNA by reverse transcription.

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 1 in NGS Workflow: Library Prep

Library preparation is crucial to the success of your NGS workflow. This step prepares DNA or RNA samples to be compatible with a sequencer. Sequencing libraries are typically created by fragmenting DNA and adding specialized adapters to both ends. In the Illumina sequencing workflow, these adapters contain complementary sequences that allow the DNA fragments to bind to the flow cell. Fragments can then be amplified and purified.

To save resources, multiple libraries can be pooled together and sequenced in the same run—a process known as multiplexing. During adapter ligation, unique index sequences, or “barcodes,” are added to each library. These barcodes are used to distinguish between the libraries during data analysis.

Library Prep Resources

Find guidance for library quantification and quality control.

Read Knowledge Article

Learn how to avoid contamination when purifying DNA/RNA.

Read Knowledge Article

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.

Jump-Start Your NGS Workflow
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Design an NGS workflow that’s right for you, process your samples, and generate your first NGS data set.

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featured NGS workflow infographic
Featured NGS Workflow

Microbial Whole-Genome Sequencing

Microbial whole-genome sequencing can be used to identify pathogens, compare genomes, and analyze antimicrobial resistance. Our featured NGS workflow for this application describes the recommended steps. The entire workflow proceeds from DNA to data in less than 24 hours.

Read Application Note

Use an extraction kit to isolate DNA from microbial colonies without introducing inhibitors. We recommend using glass beads. Assess purity using UV spectrophotometry and quantitate DNA using fluorometric methods.

Estimated time: ~1–2 hours

Prepare and quantify libraries following the protocol listed in the Illumina DNA Prep Guide. You can also perform an optional library quality check using the Agilent 2100 Bioanalyzer or Advanced Analytical Fragment Analyzer. You’ll need:

Estimated time: ~2.5 hours
Estimated DNA input: 1–500 ng

Sequence libraries in a 2 × 300 bp run following the protocol listed in the MiSeq Specifications guide . You’ll need:

Sequence libraries in a 2 × 150 bp run following the protocol listed in the iSeq 100 System Guide. You’ll need:

  iSeq 100 MiSeq
Run Time 9.5 -19 hrs 4-55 hours
Maximium Output 1.2 Gb 15 Gb
Maximum Reads Per Run 4 million 25 million
Maximum Read Length 2 x 150 bp 2 x 300 bp

† For MiSeq V3 kits only

Analyze data using the BWA Aligner app and visualize data using the Integrative Genomics Viewer app in BaseSpace Sequence Hub. You’ll need:

Estimated analysis time: ~1 hour

Learn More About Microbial Whole-Genome Sequencing

Additional Resources

Introduction to NGS

This in-depth overview describes the basics of Illumina sequencing chemistry, major advances in technology, and more.

qPCR to RNA-Seq Workflow Guidance

Explore the app note for the best practices for transitioning from qPCR to custom RNA sequencing.

Methods Guide

Access a comprehensive list of products for sequencing whole microbial genomes, mRNA, environmental samples, targeted regions, custom-selected regions, protein-binding regions, and more.

NGS Tutorial

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

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 Course
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