CleanPlex for MGI SARS-CoV-2 Panel for COVID-19 coronavirus detection, tracking, and research via amplicon-based target enrichment for Next Generation Sequencing (NGS) on MGI Tech’s DNBSEQ platforms
Paragon Genomics designed a highly multiplexed target enrichment panel covering the entire genome of SARS-CoV-2 virus (except for 92 bases at the ends). The panel enables complete genome sequencing and epidemiological studies of the new SARS-CoV-2 virus responsible for the COVID-19 pandemic. With Paragon Genomics CleanPlex technology, the entire genome of the virus can be amplified from RNA to sequence-ready libraries in 5 hours. Two versions of this the panel are available for two major sequencing platforms: Illumina and MGI. CleanPlex Technology allows the ease and flexibility to sequence samples for confident infectious disease surveillance and research.
- Significant Cost Savings
98% reduction in sequencing cost. Only 0.2M reads per sample required v.s. 10M reads required by many shotgun metagenomics sequencing methods.
- High Coverage of Target Regions
99% coverage of the entire SARS-CoV-2 genome
- Sensitive Detection
Can detect down to 1 copy with high confidence compared 3-5 copies by RT-qPCR (1)
- Fast, Streamlined Workflow
Generate sequencing-ready libraries in just 5.5 hours using a rapid, four-step protocol including RT step with minimal hands on time.
- Superb Performance
Prepare high-quality NGS libraries with excellent on-target performance using CleanPlex Technology to enable efficient use of sequencing reads. CleanPlex panels’ on-target rates are usually much higher than those of hybrid captured-based small target enrichment panels.
The CleanPlex SARS-CoV-2 Panel contains the CleanPlex Multiplex PCR Primers and CleanPlex Targeted Library Kit with RT components. CleanPlex Indexed PCR Primers and CleanMag Magnetic Beads are ordered separately to complete the workflow from input RNA to sequencing-ready NGS libraries.
(1) Corman VM et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988269/
For Research Use Only. Not for use in diagnostic procedures.
(Left) Using plasmids containing the N and S genes of SARS-CoV-2 on with a single pool workflow for detection, the detection rate at low copy numbers are show. The detection rate was shown to be 56% at 1.1 copy and 100% when more than 2 copies were present. With the use of both pools to cover the entire viral genome, the sensitivity is expected to only further increase. (Right) For these detected, the panel was able to on average capture all targets within 10 fold read depth range for 2.8 viral copies, and similarly for all but one target for 1.4 viral copies. All targets were also uniformly distributed across the GC range.
Related publication: High sensitivity detection of coronavirus SARS-CoV-2 using multiplex PCR and a multiplex-PCR-based metagenomic method
Enrichment method: Multiplex PCR
Strain compatibility: Complete coverage of major strains: MN908947 and MT007544
Cumulative target size: 29,903 bp
Number of amplicons: 343
Amplicon size: 116 – 196 bp, Median 149bp
Number of primer pools: 2
Sample input requirement: 5-11 µL of extracted total RNA or ~50ng purified total RNA
Sample types: Sputum, nasopharyngeal and oropharyngeal swabs and aspirate, tissue samples, and other methods for viral RNA sampling.
Total assay time: 5 hours
Hand on time: Less than 1 hour
Design coverage: Complete coverage (except 92 bp at the ends of the genome)
Amplicon coverage (≥50x): >95% with 300 copies viral input at 0.2M PE
Reads per sample: On-Target Aligned Reads >98% with 300 copies viral input at 0.2M PE Reads per sample
Total reads per sample: 0.2 to 0.3 M PE per sample
CleanPlex for MGI Indexed PCR Primers
|Component||Cap colour||Amount for 384 reactions
|RT Primer Mix DP||Purple Striped||1152 μl
|RT Enzyme||Blue Striped||384 μl
|RT Buffer DP||Green Striped||1920 μl
|5X mPCR Mix||Green||1536 μl
|CP Reagent Buffer||White||768 μl
|CP Digestion Reagent||Yellow||384 μl
|Stop Buffer||Red||3072 μl
|5X 2nd PCR Mix||Blue||3072 μl
|TE Buffer||Clear||16 ml
|Water (DNase and RNase-free)||Clear||16 ml
, CleanMag Magnetic Beads
, and MGIEasy Circularization Kit (provided by MGI) are ordered separately.
The MGIEasy Circularization Kit is used to generate circularized DNA libraries, which are specific to and required for sequencing on MGI’s NGS instruments. Contact your local MGI sales representative.
CleanPlex is the the most advanced PCR amplicon sequencing technology.
What is Targeted Sequencing (Resequencing) / Target Enrichment?
During Target Enrichment, the target DNA sequences are either directly amplified (amplicon or multiplex PCR-based) or captured (hybrid capture-based) and then subsequently sequenced using DNA sequencers. This process is followed by Targeted Sequencing, also known as Resequencing, where only the region of interest containing the target DNA is sequenced instead of a whole genome.
Why Target Enrichment?
Whole genome sequencing (WGS) and its corresponding whole genome amplification (WGA)’s applications are more suited for research and discovery. While Targeted Sequencing and Target Enrichment are preferred in a fast-growing clinical and industrial context where cost and speed are key.
CleanPlex NGS Target Enrichment Amplicon Sequencing Technology
CleanPlex technology is an ultra-high multiplex PCR amplicon-based target enrichment technology for NGS targeted sequencing (amplicon sequencing as opposed to hybrid capture-based sequencing). It features a highly advanced proprietary 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.
- High accuracy with lower sequencing cost: super high amplification uniformity and super low PCR background noise
- Easily automated: single-tube and 3-hour workflow with minimal hands-on time
- High compatibility: suited for 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 amplicons to over 20,000 amplicons in a single multiplex PCR pool
CleanPlex multiplex PCR-based NGS target enrichment workflow.
CleanPlex Ready-to-Use and Custom NGS Panels allow high-quality target-enriched NGS libraries to be easily and quickly prepared for amplicon sequencing.
CleanPlex background cleaning chemistry.
The CleanPlex chemistry’s streamlined protocol can be completed with 3 simple steps, each consisting of a thermal cycling or incubation reaction followed by a library purification using magnetic beads.
Step 1: targets of interest are amplified in a multiplex PCR reaction.
Step 2: primer-dimers, non-specific PCR products, and complex molecular-debris are biochemically removed in a digestion reaction.
Step 3: libraries are barcoded with sample indexes and amplified in a PCR reaction.
High performance powered by 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.
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.
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