New to Sanger and Next-Generation Sequencing Technology
I’m new to Sequencing
If you are new to sequencing, you’ve come to the right place to learn more about sequencing and determine which sequencing technologies will best fit your needs. We are a leader in sequencing technologies, with our Applied Biosystems™ genetic analyzers and Ion Torrent™ next-generation sequencing systems. Our sequencing platforms are prominent in the history of sequencing, and we strive to actively shape the future of sequencing technology.
Sequencing is the process of reading the nucleotides present in DNA or RNA molecules. There are two types of sequencing technologies that are used today: Sanger sequencing and next-generation sequencing. Each of these technologies has utility in today’s genetic analysis environment.
Sanger sequencing is the method developed by British biochemist Dr. Frederick Sanger in the 1970s. This method involves copying single-stranded DNA with chemically altered bases called dideoxynucleotides which, when incorporated at the 3' end of the growing chain, terminate the chain selectively at A, C, G, or T. The terminated chains are then resolved by capillary electrophoresis.
Next-generation sequencing (NGS) utilizes massively parallel sequencing to generate thousands of megabases of sequence information per day. Next-generation techniques are based on a "sequencing by synthesis" principle, where nucleotides incorporated into a strand of DNA provide a unique signal. In most NGS technologies, the unique signal is a fluorescent molecule. However, with Ion Torrent™ next-generation sequencing technology, the signal is in the form of a pH change, eliminating the need for light-based detection.
Video: DNA Sequencing
DNA sequencing is the process of reading nucleotide bases in a DNA molecule. During Sanger sequencing, DNA polymerases copy single-stranded DNA templates by adding nucleotides to a growing chain (extension product).
Sanger sequencing can be accomplished in a single day with the 5-step process described below.
NGS experiments can be completed in as little as 1 day, but can also take up to 1 week or more, depending on the technology and throughput.
The best sequencing platform for you will depend on the applications you plan to run, which help define the throughput, read length, and accuracy requirements for your sequencing research projects. We have profiled the key sequencing applications to help you identify the sequencers that best fit your needs.
For targeted sequencing applications that require interrogating hundreds to thousands of genes at a time, a benchtop NGS sequencer such as the Ion PGM™ System provides the ideal throughput per run, and studies can be performed quickly and cost-effectively. Targeted sequencing by NGS is now very easy to get up and running, with the availability of premade panels such as the Ion AmpliSeq™ panels. These premade panels contain the materials to isolate and sequence targets related to a certain disease, such as cancer. Custom panels can also be generated quickly and easily.
Sequencing of less than 100 target regions and confirming results from larger targeted sequencing studies is accomplished most effectively by Sanger sequencing. Sanger sequencing is the gold standard sequencing technology, so results from other sequencing platforms are usually verified using Sanger sequencing. A Sanger sequencing run can be accomplished in about 5 hours at a cost as low as a few dollars per sample, so it is an accurate, fast, and low-cost method for small targeted sequencing studies. Confirmation of NGS results is also very easy to accomplish with tools like the Primer Designer™ Tool for Sanger sequencing, which contains >300,000 predesigned primer pairs targeting the human exome.
RNA and transcriptome sequencing
NGS is highly suitable for RNA and transcriptome sequencing applications. Targeted RNA sequencing studies can be performed on a benchtop system, while transcriptome studies are best accomplished using a higher throughput system such as the Ion Proton™ System.
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Exome sequencing is a targeted sequencing approach for the approximately 1% of the genome that is expressed as genes. Exome sequencing is used to identify disease-causing variants when your research requires high throughput, accuracy, and a simple workflow. Exome sequencing can be performed with an NGS platform such as the Ion Proton™ System, which typically requires only 60 minutes of hands-on time and can run up to 24 exomes per week.
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De Novo sequencing
The initial generation of the primary genetic sequence of a particular organism is called de novo sequencing. De novo sequencing is best accomplished using long read lengths to provide the best coverage of the genome. The 400 base pair read lengths provided by the Ion PGM™ Sequencer allow de novo sequencing of genomes. Sanger sequencing provides longer and more accurate read lengths than NGS technologies do; however, the low throughput scale is not ideal for de novo sequencing. Sanger sequencing is useful for filling in regions that are too difficult to sequence by NGS and closing gaps in NGS-generated de novo sequences.
Fragment analysis is the term for applications such as STR analysis that rely on size separation for analysis of DNA fragments. Fragment analysis applications can only be run on a high-resolution capillary electrophoresis instrument such as the Applied Biosystems™ genetic analyzers used for Sanger sequencing.
Exome sequencing is a targeted sequencing approach for the approximately 1% of the genome that is expressed as genes. Exome sequencing is used to identify disease-causing variants, requiring high throughput, accuracy, and simple workflow. Exome sequencing can be performed with an NGS platform such as the Ion Proton® System, which requires only 60 minutes of hands-on time and can run up to 24 exomes per week.
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Learn more about our sequencing applications:
- Behind the Bench sequencing blog
- Technical support
- Capillary Electrophoresis Software Support Center
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