The most advanced NGS amplicon sequencing technology

CleanPlex® NGS Amplicon Sequencing Technology

CleanPlex® technology is an ultra-high amplicon-based target enrichment technology for targeted sequencing (amplicon sequencing as opposed to hybrid capture-based sequencing). It features a highly advanced proprietary multiplex PCR primer design algorithm, an exceptionally uniform multiplex PCR amplification chemistry and an innovative, patented background cleaning chemistry. Together, they allow CleanPlex Ready-to-Use and Custom NGS Panels to break the limits of existing PCR amplicon-based and hybrid capture-based target enrichment technologies.


Feature Highlights:

  • Super high amplification uniformity and super low PCR background noise (more accurate variant calling or less sequencing cost)
  • Single-tube and 3-hour workflow with minimal hands-on time (easy automation)
  • Compatible with difficult samples (degraded FFPE, cfDNA) and major sequencing platforms (Illumina, Ion Torrent, MGISeq)
  • Extreme sensitivity (down to single cell level direct amplification*)
  • Excellent panel size scalability from a few to over 20,000 amplicons in a single multiplex PCR pool

*based on data from our collaborator RareCyte

Below is a high-level targeted sequencing workflow (Figure 1) starting from DNA extraction to sequencing data analysis. The target enrichment and library preparation workflow (Step 2) is critical and often times determines the quality of the sequencing data.

CleanPlex targeted DNA sequencing workflow

Figure 1. CleanPlex Targeted DNA Sequencing Workflow

Below is a typical workflow (Figure 2) of CleanPlex DNA target enrichment and library preparation. The CleanPlex DNA workflow involves 3 simple steps, each consisting of a thermal cycling or incubation reaction followed by a library purification using magnetic beads. The streamlined protocol can be completed in just 3 hours. In step 1, targets of interest are amplified in a multiplex PCR reaction. In step 2. primer-dimers, non-specific PCR products, and complex molecular-debris are biochemically removed in a digestion reaction featuring the innovative and proprietary CleanPlex Background Cleaning chemistry. In step 3, libraries are barcoded with sample indexes via a PCR indexing reaction.The input materials could be genomics DNA, DNA extracted from FFPE or fresh/frozen tissues or blood liquid biopsies. By adding a reverse transcription step in the front, the input material could be RNA too. (Figure 3)

cleanplex DNA amplicon sequencing target enrichment and library preparation workflow

Figure 2. CleanPlex DNA Target Enrichment and Library Preparation Workflow

cleanplex RNA target enrichment library preparation workflow

Figure 3. CleanPlex RNA Target Enrichment and Library Preparation Workflow

High performance amplicon sequencing powered by PCR background cleaning

Non-specific PCR products and primer-dimers are biochemically removed using the proprietary CleanPlex digestion chemistry. This ensures that only DNA sequences of interest are converted into NGS library molecules, resulting in highly efficient use of sequencing reads.

CleanPlex background removal library trace for amplicon sequencing

Effective removal of PCR background. Libraries were prepared using the CleanPlex OncoZoom Cancer Hotspot Panel with (blue trace) or without (red trace) using the CleanPlex digestion reagent and examined using an Agilent® Bioanalyzer®. Without CleanPlex digestion, significant PCR background was formed, which would result in low mapping rate and poor on-target rate and require more sequencing reads to obtain adequate data. With CleanPlex digestion, nearly no background was generated, producing a sharp and clean library peak in the Bioanalyzer trace. The proprietary CleanPlex digestion chemistry is essential for removing undesired side products formed during multiplex PCR  amplification of target sequences.

Interrogate more targets without performance compromise 

CleanPlex® Ready-to-Use and Custom NGS Panels are designed using our proprietary ParagonDesigner algorithm and optimized through an iterative process by our expert scientists to ensure maximum target design rate, as well as robust coverage uniformity, high on-target rate, and low amplification biasMore than 20,000 amplicons per primer pool can be multiplexed in a single CleanPlex reaction. Amplicon size can be tuned to specification to ensures high performance with the desired sample type. 

cleanplex amplicon panel scalability

Effective background removal for all multiplexing levels. Libraries were prepared with CleanPlex NGS Panels of varying sizes (7 to 8,000 amplicons) and examined on an Agilent® Bioanalyzer®. All libraries generated a clean peak indicating minimal formation of non-specific PCR products. The data shows that CleanPlex background cleaning chemistry is effective regardless of the level of multiplexing, which means new targets can be added without affecting performance.

cleanplex_mapping_rate_and_on_target_rate_uniformity

High performance regardless of panel size. Libraries were prepared with CleanPlex NGS Panels of varying sizes (15 to 1,500 amplicons) and sequenced on an Illumina® platform. Mapping rate was maintained above 96% and increased with the number of amplicons used per reaction, indicating that CleanPlex chemistry is even more effective on libraries prepared using larger numbers of amplicons. On-target rate was also higher than 96% for all panel sizes. Coverage uniformity, measured as % covered by at least 0.2X mean coverage, was maintained above 96%. 

High quality amplicon libraries from low-input and challenging samples

CleanPlex®‘s streamlined workflow minimizes sample loss to preserve genomic information in low-input and challenging samples, such as degraded FFPE DNA from tumor samples, cell-free DNA from liquid biopsies and single circulating tumor cell (CTC) from blood. High quality target-enriched libraries can be generated from as little as 1 ng of DNA and even down to 6 pg of DNA from a single CTC.

cleanplex_coverage_uniformity_under_different_DNA_input

High performance even with low input amounts. Libraries were prepared with the CleanPlex OncoZoom Cancer Hotspot Panel (601 amplicons) using varying amount of input DNA and sequenced on an Illumina platform. CleanPlex technology was able to produce libraries with >95% coverage uniformity (measured as % covered by at least 0.2X mean coverage) using as little as 50 pg of input genomic DNA.

Single cell targeted DNA sequencing with CleanPlex OncoZoom Cancer Hotspot Panel

CleanPlex NGS Panels can directly amplify single cells without pre-amplification such as whole genome amplification (WGA) while still maintaining high amplification uniformity. In addition, this avoids WGA bias and has much lower false positives and false negatives in terms of variant calls.

Single Single cell targeted DNA sequencing with CleanPlex OncoZoom. Single cell lysate obtained from RareCyte’s RareCyte’s CTC assays and CyteFinder Instrument is input as template into Paragon’s CleanPlex OncoZoom Cancer Hotspot 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.

Detect variants with high confidence (without unique molecular identifiers – UMIs)

CleanPlex histogram of error allele frequency

High quality sequencing data with low error rate. A library was prepared with the CleanPlex OncoZoom Cancer Hotspot Panel (610 amplicons) using 10 ng of Horizon Discovery HD780 cfDNA reference standard and sequenced on an Illumina platform to an average read depth of 8,500. A histogram of the frequency of random errors from sequencing shows that CleanPlex generated high quality data with the majority of random errors present at less than 0.2% frequency. This low level of background errors allows confident variant calling of mutations at 1% allele frequency.

CleanPlex variant calling concordance

High variant call concordance. Libraries were prepared with the CleanPlex OncoZoom Cancer Hotspot Panel (610 amplicons) using 10 ng of Horizon Discovery HD780 cfDNA reference standards and sequenced on an Illumina platform to an average depths ranging from around 1,100 to 9,800 reads per amplicon. The data shows that CleanPlex consistently detected validated variants at the expected frequency. The red lines represent the known allele frequencies of the eight mutations, and numbers are the averages of the detected frequencies for each allele with standard deviation error bars.

Input to target-enriched NGS libraries in a single-tube in 3 hours

CleanPlex® NGS Panels feature a rapid, single-tube workflow* that can be completed in 3 hours and requires only 75 min of hands-on time. High-quality target-enrich libraries can be easily and quickly prepared for faster time to results.

Single-tube workflow minimizes sample loss to preserve genomic information in the sample, and reduces the likelihood of errors and sample mix ups to ensure positive sample identification.

* Single-tube workflow. A single-tube workflow is followed when using a single-pool CleanPlex NGS Panel. For a multi-pool CleanPlex NGS Panel, the individual mPCR products from the primer pool-specific reactions are combined into one tube, and the remaining protocol is carried out using a single-tube workflow.

Simple workflow translates to high reproducibility

CleanPlex NGS target enrichment library prep workflow reproducibility

High reproducibility. Eighteen CleanPlex libraries were generated by 3 operators and sequenced on an Illumina platform. The GC coverage of the libraries were plotted and compared against each other. The libraries produced highly reproducible GC coverage profiles, yielding a Pearson correlation coefficient of 97.26% ± 1.07%.

Reduce sequencing cost with high quality libraries

The combination of low PCR background, low GC bias, and high mapping rate, on-target rate, and coverage uniformity means that very few sequencing reads are wasted on sequencing off-target sequences and non-specific PCR products and primer-dimers. It also means that fewer sequencing reads are required to ensure all targets are covered at a minimally required depth to make confident base calls. Overall, CleanPlex® NGS Panels allow efficient use of sequencing reads so that sequencing can be performed cost-effectively by allowing more samples to be sequenced at a time.

CleanPlex amplicon sequencing uniformity and cost

High performance translates to cost-effective sequencing. A 207-amplicon panel was used to generate target-enriched NGS libraries using either the CleanPlex or Competitor T’s library preparation chemistry. Libraries were sequenced and analyzed for coverage uniformity across GC content. The results indicate that for a 207-amplicon panel, 60% less sequencing would be required using CleanPlex, which means 2.5X more samples can be sequenced on a flow cell. To achieve similar data quality, CleanPlex’s mean read depth could be reduced to 600X coverage while Competitor T’s would need to be increased to >1,500X coverage.

Discover more with less™

The combination of superior primer design and innovative library preparation chemistry give rise to CleanPlex NGS Target Enrichment 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 

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

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

View more data on CleanPlex® technology for amplicon sequencing

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