Overview of Next-Generation Sequencing (an advanced version of DNA sequencing)
Next-Generation Sequencing (NGS) is a massively parallel method to sequence thousands to millions of DNA molecules simultaneously. Since NGS was first introduced in 2005, it has risen to become the predominant DNA sequencing technology and heralded new levels of accessibility for research, industry, and even everyday consumers alike. Most importantly, it has begun to make precision medicine a reality, with clinicians able to sequence a patient’s whole genome, exome or a targeted gene panel cheaply, and prescribe therapies tailored to individual genetic profiles.
Improvements in DNA sequencing technologies since 1990 have resulted in dramatic reductions in the time and cost of sequencing a single human genome. Today, NGS technology can sequence a human whole genome (more than 3 billion basepairs) within two days at a dramatically lower cost.
What is Targeted Sequencing (Resequencing) via Target Enrichment?
Targeted Sequencing or Resequencing is a method for only sequencing part of a whole genome or regions of interest without sequencing the entire genome of a sample. In order to only focus on specific or clinically relevant regions of a genome or DNA sample, it requires a pre-sequencing DNA preparation step called Target Enrichment where target DNA sequences are either directly amplified (amplicon or multiplex PCR-based) or captured (hybrid capture-based) and then subsequently sequenced using DNA sequencers. Below is a typical targeted sequencing workflow. (Figure 1)
Figure 1. A Typical Targeted DNA Sequencing Workflow
Why Target Enrichment?
Whole genome sequencing (WGS) and its corresponding whole genome amplification (WGA) method are more suitable for research and discovery types of applications, while targeted sequencing and target enrichment are essential to many fast-growing clinical and industrial applications where cost and speed are more important.
One of the challenges the genomics community faces is the continued acquisition of large amounts of sequencing raw data that is yet to be fully and successfully translated and interpreted to help advance research, diagnose and cure diseases on a wider scale. Even with current reduced sequencing costs, a whole genome sequencing approach can be practically used only in specific scenarios such as basic research, population genetics or rare disease detection. A focused or targeted approach would be more appropriate for understanding disease progression and guiding therapy selection in clinical setting or massively screening DNA samples in industrial applications. In addition, sequencing an entire genome or exome can be prohibitively expensive in terms of laboratory operations and bioinformatics infrastructure for storing and processing large amounts of data. Therefore, target enrichment has become vital for the continued progress of precision medicine and research.
The following table compares WGS and targeted sequencing in terms of sequencing and library preparation reagent costs. In addition to reagent costs, bioinformatics analysis time and cost shall also be taken into account when considering the suitable methods for your applications.
|Comparison of WGS and Targeted Sequencing Methods|
|Human Whole Genome||Human Whole Exome (50Mb)||Targeted Panel (1Mb)|
|Target Region Size (basepair)||3 x 109||5 x 107||1 x 106|
|Depth of Coverage||30X||100X||1000X|
|Number of Samples per Sequencing Run*||8||145||725|
|Cost of Sequencing Reagent per Sample**||$1,500||$100||$15|
|Cost of Target Enrichment per Sample***||N/A||$250||$100|
|Total Cost per Sample (excluding bioinformatics)||$1,500||$350||$115|
* Based on Illumina HiSeq 2500 System dual flow cell high output mode
** Based on Illumina HiSeq SBS V4 cost
*** Based on average target enrichment and library preparation kit prices
The clear benefits of targeted resequencing are driving the adoption of NGS in areas such as translational research, clinical diagnostics and industrial applications while the value also shifts from sequencing reagents to target enrichment reagents on a per sample basis. For a small targeted sequencing panel covering for example only 100 kb region of interest, most of the cost is incurred at the library preparation and target enrichment steps while the cost of sequencing reagents is negligible for each given sample if deep sequencing is not required.
What are major applications of Targeted Sequencing and Target Enrichment?
Currently, targeted sequencing and target enrichment have been applied to many areas from basic research to clinical diagnostics and applied markets. Below is a list of major applications.
- Cancer research and diagnostics
- Tumor profiling
- Cancer liquid biopsy
- Minimal residual disease testing
- Circulating Tumor Cell (CTC) analysis
- Reproductive Health
- Carrier screening
- Non-invasive prenatal testing
- Preimplantation genetic diagnosis (PGD)
- Newborn screening
- Cardiovascular Disease Testing
- Infectious Disease Testing and Surveillance
- Transplant Genomics / HLA typing
- Inherited Disease Testing
- Metabolic and Immune Disorders
- Neurological Disease Testing
- Companion Diagnostics
- Applied / Industrial Applications
- Agrigenomics / molecular breeding
- Food safety and animal health
- Environmental research
Discover more with less™
The combination of superior primer design and innovative multiplex PCR-based target enrichment and library preparation chemistry give rise to CleanPlex NGS Amplicon Panels’ ultra-high multiplexing capability, high performance, low input requirement, high sensitivity, single-tube workflow, and cost-effective amplicon sequencing. These remarkable features and benefits allow researchers and assay developers to discover more with less.
Discover more with CleanPlex NGS Panels
• Multiplex 20,000+ amplicons per reaction
• High target design rate
• High coverage uniformity
• High on-target rate
• High sensitivity (1% LOD with 10 ng input)
Use less input and resources to reduce costs
• Inputs as low as 10 ng
• Fast 3-hour protocol
• Simple, streamlined workflow
• Efficient use of NGS reads