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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

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.

View Bulletin

Learn how to avoid contamination when purifying DNA/RNA.

View Bulletin

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
Jump-Start Your NGS Workflow

Want to get started faster? We offer services to help you 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

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

Estimated run time: ~19.5 hours
Estimated output: 1.2 Gb per 2 × 150 bp run
Samples per run: 4–5 samples; assuming 5 Mb at 50× coverage

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

Bacterial and Viral Small Whole Genome Sequencing with iSeq 100

Guided NGS Tutorials

Learn what to expect with these tips for each step of the NGS workflow.

View Tutorials

Additional Resources

Sequencing: Illumina Technology

A general overview of the Illumina sequencing workflow, from DNA/RNA extraction to the completion of a run.

In-Depth Introduction to NGS

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

qPCR to RNA-Seq Workflow Guidance

Best practices for transitioning from qPCR to custom RNA sequencing.