NGS Target Enrichment and Amplicon Sequencing
The two most commonly used techniques for NGS target enrichment are multiplex PCR-based (amplicon sequencing) and hybrid capture-based enrichment.
In general, amplicon sequencing method is faster, easier and cheaper than hybridization-based alternatives and so more suited for quick gene panel testing and production-scale applications such as clinical diagnostics and industrial genomic screening. Amplicon sequencing also allows for dealing with low DNA input which is difficult for hybrid captured based technology. For example, when dealing with viral detection, amplicon sequencing has a clear advantage against hybrid capture based methods in terms of detection sensitivity and DNA/RNA input requirements.
On the other hand, hybrid capture-based method can interrogate very large target region up to human whole exome and is generally better at detecting structural variations such as novel gene fusions, etc., so it is more suitable for research and discovery projects. However, capture-based method suffers low on-target rate on smaller panels due to its inherent lower specificity of hybridization probes. Due to the above reasons, scientists and clinicians tend to employ multiplex PCR for small (less than 1Mb target region) or hotspot panels for the detection of single nucleotide polymorphisms (SNPs) and/or small insertions/deletions and hybrid capture for large (0.5Mb – 50Mb target region) panels for the detection of SNPs, fusion genes, copy number variations (CNV), etc.
Below is a comparison of typical workflows of Amplicon-based target enrichment and hybrid capture-based target enrichment.
A Typical Amplicon Sequencing Library Preparation Workflow
A Typical Hybrid Capture Target Enrichment Workflow
Below is a summary of the pros and cons of both methods.
Note: Some comments on Amplicon-based target enrichment do not apply to Paragon Genomics’ CleanPlex technology as it overcomes many of the drawbacks of traditional amplicon-based methods. This will be discussed in the next section.
|Traditional Amplicon Sequencing Technologies||Hybrid Capture-based Target Enrichment Technologies|
|Requires lower input of DNA (1-10 ng)||Can support large panel size (up to human whole exome)|
|Shorter and easy workflow (3-6 hours)||Can detect some novel structural variations such as novel gene fusions|
|High on-target rate (>95%)||has better assay uniformity in general|
|No special equipment required (e.g. no need of DNA fragmentation)||Custom assay probes are easier to design|
|Better performance on difficult clinical samples such as FFPE tissue DNA|
|Lower reagent and consumable (pipette tips) costs|
|PCR amplification bias resulting in lower assay uniformity, especially evident for large panels||Needs to fragment DNA first|
|Non-specific PCR background noise (e.g. primer dimer) can be high, especially evident for large panels involving over 1,000 amplicons in a single pool||Requires higher input DNA (>10 ng)|
|Difficult to design a large number of multiplex PCR primer pairs that are compatible in a single pool with minimal interaction||Longer and laborious workflow (>10 hours)|
|Low on-target rate for small panels and mixed samples (e.g. detection of bacteria or viruses in human DNA background); some as low as 10-20%|
|Higher reagent and consumable (pipette tips) costs|
|Novel fusion detection (cannot design primers for unknown fusion breakpoints)||TCR/BCR sequencing (Application doesn’t allow for fragmenting genomic DNA due to the need to preserve TCR/BCR variable regions’ integrity)|
|Whole Exome Sequencing (target region can be too large and uniformity will suffer)||Pathogen detection on mixed samples with varying DNA inputs (e.g. TB patient samples)|
CleanPlex® Amplicon Sequencing Technology – a novel ultra-high multiplex PCR-based target enrichment method that bridges the gap between traditional amplicon sequencing and hybrid capture
Overcoming Background Noise
First, the targeted regions of a genome or DNA sample are amplified by well-designed multiplex PCR primers with overhanging tails being partial adaptor sequences compatible with corresponding DNA sequencers, resulting in both target amplicons and non-specific PCR products including primer dimers.
Traditionally, when the panel size is large (e.g. more than 1,000 amplicons in a single pool), the non-specific PCR products can be overwhelming and significantly affect the downstream steps if there is no measure to remove them. Some amplicon-based methods utilize bead purification and size selection to remove smaller DNA fragments such as primer dimers. However, some complicated non-specific PCR products with sizes similar to the lengths of target amplicons and its resulting libraries can be difficult to remove using just size selection. The following Bioanalyzer trace shows significant background noise around a target library of 300bp.
CleanPlex overcomes this drawback with an innovative and patented enzymatic background cleaning step that removes non-specific PCR products including both primer dimers and more complicated and longer nonspecific PCR artifacts, resulting in very pure target libraries. The following Bioanalyzer trace shows the effect of CleanPlex background cleaning technology.
Subsequently, sample barcodes (for sample pooling purpose) are added by an indexing PCR step to get sequencing-ready libraries. The whole workflow only takes 3 hours and minimal hands-on time.
Overcoming Scalability or Panel Size Limitation
Due to its background cleaning technology, CleanPlex can easily break the panel size limit of traditional multiplex PCR and amplify more than 20,000 targets in a single panel. The following amplification plot shows the GC% of each target amplicon vs its sequencing depth for a 27,000-amplicon panel.
Overcoming GC Bias and Uniformity Issues
The figure below compares CleanPlex and another well-known amplicon-based method in terms of GC bias and amplification uniformity. Two panels target the same regions of a few cancer-related genes. The competitor method obviously has GC bias around low GC region and an overall low amplification uniformity across the spectrum of the panel. On the contrary, CleanPlex can amplify all target regions evenly. The result of this is that users of the competitor’s panel have to raise the average sequencing depth by 100% to achieve similar variant calling quality, therefore doubling the sequencing depth and cost required for CleanPlex panel.
Pushing the limit of LOD
Our partner RareCyte, Inc has successfully demonstrated that CleanPlex technology can directly amplify DNA from a single cell (only ~6 pg of DNA), specifically circulating tumor cells isolated from cancer patient blood.
Single cell lysate is input as template into Paragon Genomics’ CleanPlex OncoZoom Panel, with modified primer concentration, PCR cycle number, and clean-up steps to compensate for low input DNA concentration. Using this non-WGA method vastly improves: (A) coverage uniformity, and incidence of (B) false negative and false positive errors, when compared to single cell WGA products.
Overcoming limitation on Detecting Novel Fusion Genes
CleanPlex Fusion Detection Technology leverages template switching and single primer amplification methods to detect novel fusion genes.
In summary, CleanPlex Amplicon Sequencing Technology leverages the speed, sensitivity, workflow and cost advantages of multiplex PCR for NGS target enrichment while overcoming its key drawbacks, therefore bridging the gap between traditional amplicon-based target enrichment method and hybrid captured-based approach.
|Traditional Amplicon- or PCR-based|
|Requires lower input of DNA (1-10 ng)||CleanPlex can directly amplify single cell DNA (6 pg)|
|Shorter and easy workflow (3-6 hours)||Advantages maintained|
|High on-target rate (>95%)||Advantages maintained|
|No special equipment required (e.g. no need of DNA fragmentation)||Advantages maintained|
|Better performance on difficult clinical samples such as FFPE tissue DNA||Advantages maintained|
|Lower reagent and consumable (pipette tips) costs||Advantages maintained|
|PCR amplification bias resulting in lower assay uniformity, especially evident for large panels||Can achieve more than 95% uniformity (0.2x) even for large panels involving over 20,000 amplicons|
|Non-specific PCR background noise (e.g. primer dimer) can be high, especially evident for large panels involving over 1,000 amplicons in a single pool||Proprietary CleanPlex background cleaning technology can effectively remove background noise regardless of panel sizes|
|Difficult to design a large number of multiplex PCR primer pairs that are compatible in a single pool with minimal interaction||ParagonDesigner™ algorithm takes into account multiple primer design factors and ensures best performance for even very large panels|
|Novel fusion detection (cannot design primers for unknown fusion breakpoints)||CleanPlex OmniFusion single primer technology enables detection of novel fusions|
|Whole Exome Sequencing (target region can be too large and uniformity will suffer)||CleanPlex is the most scalable and uniform multiplex-PCR based technology and will be able to amplify whole exome.|
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