|Compared to other target enrichment methods such as hybrid capture or metagenomics, amplicon-based target enrichment has shown to exhibit higher sensitivity with lower read depth requirements for the same coverage, especially with low copy number inputs. Regardless of your application — strain confirmation, mutation detection, phylogenetic research, or intra-host variant calling — our amplicon based solution achieves the highest ratios of coverage and sensitivity to sequencing read depth.|
|The <100% coverage is strictly due to amplicon-based amplification methods. The uncovered regions are the 92 basepairs at the very beginning and very end of the viral genome. Because targeted sequencing requires primer pairs for amplification, it’s not possible to design primers for a region that ends, therefore the ends of the genome (both ends) are not covered. This is just a negligible fraction of the entire genome and will not impact your applications in detection, surveillance, phytogenetic studies, etc.|
After your order is shipped, you will be able to download the BED files from My Downloads using the account associated with the order.
|As of now, our SARS-CoV-2 panel is available for Illumina and MGI sequencing. Currently, we are working on optimizing the protocol for Ion Torrent sequencing platforms.|
|Our digestion step is used to remove non-specific products formed during the initial mPCR step. Background noise is common in approaches using an mPCR step, however our novel approach to dealing with these off-target products is specific to our protocol, and allows us to obtain cleaner final libraries than our competitors.|
|The number of samples multiplexed per run depends on customer applications, level of coverage needed, and sequencing capacity used. To obtain sufficient coverage for genotyping, we recommend 100x cluster reads per amplicon. For sufficient coverage of samples for surveillance and low variant calling, we recommend 1000X cluster reads per amplicon. The full panel contains 343 amplicons, meaning approximately 70K PE reads and 700K PE reads per sample for genotyping and low frequency variant calling respectively. Depending on your sequencer’s capacity, you can calculate the number of samples per run.|
|For Illumina, we offer combinatorial index options at 8, 192, 384, and 2688 combinations for sample multiplexing per run. Additionally, we offer an option of 32 Unique Dual Indexes (UDI) and are quickly working towards 384 UDIs that will be available soon.|
For MGI single-indexes primers, we currently offer 96 indexing options in sets of 16.
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|Because our amplicon length ranges between 116-196 bp ( amplicon insert + primer), to obtain full coverage we recommend 2x 150bp sequencing. The panel can also be run with 2x 75 bp sequencing, however not all bases will be covered. If your application does not require full coverage and quick sequencing turn around time is more important, 2 x 75 bp is compatible with our products and can be both cost saving and time efficient.|
|Because we have 343 pairs of strategically designed primers, coupled with the already established CleanPlex Technology, our panel enables amplification of extremely low quality RNA samples. In addition to lower quality RNA tolerance, our kit also demonstrates great sensitivity to viral mutations, and enables confident variant calling.|
|For an internal positive control to ensure library prep was performed correctly, we have human RNA targets as internal controls available upon request. For positive SARS-COV-2 controls, there are many types of positive sample controls available (https://www.amp.org/clinical-practice/testing-resources-for-covid-19/) to fit your application needs and we do not recommend a specific product.|
|Our SARS-COV-2 Panels workflow is compatible with total RNA, total nucleic acid, and viral specific RNA extraction kits. The most important aspect is that the viral RNA is present in the extracted product. Not all extraction kits are created equal; we suggest testing the extraction methods to ensure your method is effective with good recovery. Because our panel is viral RNA specific, using total nucleic acid (DNA+RNA) as a starting point does not pose any issues to our chemistry and there is no need to perform DNA removal with DNAse.|
Some compatible kits include, but are not limited to, QIAamp® Viral RNA Mini Kit, QIAamp® MinElute Virus Spin Kit, RNeasy® Mini Kit (QIAGEN), EZ1 DSP Virus Kit (QIAGEN), Roche MagNA Pure Compact RNA Isolation Kit, Roche MagNA Pure Compact Nucleic Acid Isolation Kit, Roche MagNA Pure 96 DNA and Viral NA Small Volume Kit.
|The CleanPlex SARS-COV-2 panel reagent kit contains reverse transcription reagents to take RNA as input. However, if you’re starting with cDNA as your input, you can skip directly to the mPCR step. This may require cDNA input amount and 2nd PCR cycle titrations to ensure optimal library yield. Ensure the cDNA input material does not contain any PCR inhibiting components that will negatively affect the mPCR reaction.|
|The primers are designed to tile across the entire genome for full genome coverage. Because the primers overlap in the process, every other primer is separated into a separate pool such that the overlapping primers do not form primer dimers. The two pool design also allows detection using only one pool at once ( 50% coverage) to minimize the amount of sequencing per sample, allowing increased sample multiplexing per run.|
|Either of the pools can be used individually for detection purposes if the whole genome coverage is not required. Each pool covers 50% of the genome and is evenly distributed across the SARS-COV-2 genome. Even with half coverage, the panel can still allow for low copy sensitivity and mutation detection with high confidence. Using one pool allows less sequencing per sample and thus higher sample multiplexing per sequencing run.|
|Unfortunately not. A 96 reaction kit provides 192 reactions worth of mPCR reagent for individual mPCR reactions per pool. However, the full coverage workflow includes pooling the two mPCR products to be carried through the remainder of the workflow as a single pool. This means that the reagents available for CP Digestion and 2nd PCR in a 96 reaction kit cannot support 192 reactions, as only 96 reactions worth of those reagents are provided. However the single pool workflow allows for shorter mPCR hands-on time and reduced sequencing costs per sample.|
|While knowing your RNA concentration is important, it is not necessary for our workflow. When working with samples of unknown viral concentration (as in most cases), we recommend using 11 uL of RNA volume as input for the reverse transcription step to allow for maximum copies input. When working with control viral DNA, 50ng of Human RNA can be added as background for more realistic samples testing conditions.|
|Nope! The beads are expected to be carried over from each step to the final library. Once the Beads are washed and re-suspended in TE Buffer, all of the product is released from the beads and “available” for the next steps of the protocol. We developed this method to minimize tube-to-tube transfer, allowing for maximum sample retention, so the beads do not interfere with the final product generation in any way.|
|Negative samples will not have the easily distinguishable characteristic target-specific peaks at 280bp for Illumina and 230bp f0r MGI panels. In addition, negative samples might exhibit large adapter dimer peaks due to the lack of template available in solution, causing a bias in dimer formation. These dimer peaks will occur around 180 bps for Illumina panel and around 100bp for MGI panels, which can easily be identified apart from the positive SARS-CoV-2 peak.|