CleanPlex amplicon sequencing technology for targeted DNA and RNA-Seq
CleanPlex® is an ultra-scalable and ultra-sensitive NGS amplicon sequencing technology. 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 traditional amplicon-based and hybrid capture-based target enrichment technologies.
- 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 DNA, FFPE RNA, cfDNA, cfRNA) and major sequencing platforms (Illumina, Ion Torrent, Genapsys, MGI DNBSeq)
- 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
- Detection of and analysis for single nucleotide variants (SNVs), small insertions and deletions (Indels), copy number variations (CNVs), gene fusions / splice variants, gene expression level, tumor mutational burden (TMB), microsatellite instability (MSI), internal tandem duplication (ITD), etc.
*based on data from our collaborators RareCyte and Mayo Clinic
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.
Figure 1. Targeted DNA Sequencing Workflow
Below is a typical workflow (Figure 2) of CleanPlex DNA amplicon sequencing library prep. 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 genomic DNA and DNA extracted from FFPE, 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)
Figure 2. CleanPlex DNA Target Enrichment and Library Preparation Workflow
Figure 3. CleanPlex RNA Target Enrichment and Library Preparation Workflow
View data on CleanPlex® technology for amplicon sequencing
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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.
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 bias. More 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.
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.
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%.
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.
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.
Direct single cell targeted DNA sequencing
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 cell targeted DNA sequencing with CleanPlex OncoZoom Cancer Hospot Panel. Single cell lysate obtained from RareCyte’s CTC assays and input as template into Paragon’s CleanPlex OncoZoom Cancer Hotspot Panel. 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 (even without unique molecular identifiers – UMIs)
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.
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.
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
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%.
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.
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 prep 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.
Sophia Genetics has compared CleanPlex technology with three other major amplicon-based technologies and published the results at the AMP (Association for Molecular Pathology) 2018 Annual Meeting. The following amplification plots show that CleanPlex has the best amplification uniformity (all amplicons’ read depths fall within 20% an 500% of mean depth while other technologies cannot achieve this). Besides amplification uniformity, Sophia Genetics also concluded that CleanPlex has high library conversion rate and reproducibility even on bad quality FFPE samples.