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
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.
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.
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.
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.
Want to get started faster? Consult with experimental design experts through our Workflow Design and Evaluation Service.* We’ll 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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Microbial whole-genome sequencing can be used to identify pathogens, compare genomes, and analyze antimicrobial resistance. Our featured NGS workflow describes the recommended methods and products for each step of this application.
Use an extraction kit to isolate DNA from microbial colonies without introducing inhibitors, such as EDTA or phenol:chloroform. Assess purity using UV spectrophotometry and quantitate DNA using fluorometric methods.
Prepare and quantify libraries following the protocol listed in the Nextera DNA Flex Guide. You can also perform an optional library quality check using the Agilent 2100 Bioanalyzer or Advanced Analytical Fragment Analyzer. You’ll need:
Estimated hands-on time: 3.5 hours
Estimated DNA input: 1-500 ng
Analyze data using the SRST2 App in BaseSpace Sequence Hub. This app reports the presence of sequence types from a multilocus sequence typing (MLST) database and reference genes from sequence databases. You can specify which MLST and/or sequence databases should be used to analyze your samples. You’ll need:Learn More About Microbial Whole-Genome Sequencing
A general overview of the Illumina sequencing workflow, from DNA/RNA extraction to the completion of a run.
This detailed overview describes major advances in technology, the basics of Illumina sequencing chemistry, and more.
Best practices for transitioning from qPCR to custom RNA sequencing.