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ConfirmPCR

1 Million Best-In Class PCR assays that covers all human canonical exons 
of protein coding genes. 


Easy-to-use database that contains highly versatile assays which can be used for

  • WGS/WES variant confirmation 
    10,000 validated in variant confirmation studies.
  • NGS Gap filling 
    Enrich low coverage regions.
  • Targeted resequencing with Sanger or NGS 
    54 validated gene panels totalling up to 223 genes and 4008 ConfirmPCR assays. 

All our assays have been throughly validated in silico, resulting in an off-the-shelf PCR succes rate of >97%. 
The optimized design parameters allow universal PCR conditions while ensuring uniform sequencing coverage. 

Delivered within 2-3 business in Europe & North America.


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Assay Specificity

In our paper "Single-nucleotide polymorphisms and other mismatches reduce performance of quantitative PCR assays" (Clinical Chemistry, 2013  ), we determined the impact of nucleotide mismatches in primer annealing sites on PCR efficiency. 

Various features such as single nucleotide polymorphisms (SNP) and secondary structures in primer annealing sites, are known to have a negative effect on amplification efficiency (see "Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR" by Boyle et al - 2009  - "Rapid detection of VHL exon deletions using real-time quantitative PCR" by Hoebeeck et al. - 2005  and "Single-nucleotide polymorphisms and other mismatches reduce performance of quantitative PCR assays" by Lefever et al. - 2013 ). Exclusion of these features from primer annealing sites is essential, since these can hamper proper hybridization of a primer to its target sequence. This may result in allelic drop out and reduced amplification. 

In addition, assay aspecificity titrates essential components of the PCR mix (e.g. dNTPs) away from the target of interest, resulting in an underrepresentation of the specific amplicon in the end product and potentially leading to non-equimolar end concentrations across PCR reactions requiring normalization prior to library preparation and sequencing.

Our state-of-the-art primer design pipeline takes into account all this and tries to generate assays having as little of these features present in their primer annealing site. Over 94% of our primers are free of SNPs, while for the remaining assays (5.99%), the number of SNPs is limited to one in over 92% of the assays (Figure 1). In these latter cases, the SNPs are located outside the last 5 nucleotides (3’ end) in 85% of the assays to further minimize the effect on amplification efficiency. 



As an integral part of our primer design engine, the potential of each candidate assay to generate non-specific (off-target) products, is assessed by aligning the primer sequences to the genome or transcriptome while allowing up to three mismatches per primer annealing site (and taking into account the maximum allowed product length).

By collecting all possible annealing sites, both perfect and imperfect (non-specific) ones 
+ tracking the position of mismatches in them, a score depicting the amplification potential of each site can be calculated. 

This strategy enables the selection of the best assays - i.e. guaranteeing no or minimal off-target amplification - and allows our customers to critically appreciate the quality of a ConfirmPCR assay before using it in the lab.



 The validity of our in silico specificity score, implemented in our state-of-the-art primer design pipeline, was empirically proven by evaluating almost 2300 assays in two DNA samples (for more details, see the paper in the knowledge center of our website). 

Following standard PCR, the resulting amplicons were sequenced and their on- and off-target coverage determined. The large majority of the assays (88%) demonstrated less than 2% off-target coverage (Figure 1). In addition, a good correlation could be shown between the calculated specificity score (ranging from 1 to 7, low to high degree of specificity) and the percentage of assays having more than 2% aspecific coverage (Figure 2), indicating that assays with higher specificity scores tend to result in less aspecific coverage. Together, these data illustrate once more the quality of our assays and the strength of our in silico specificity assessment workflow and primer design pipeline.



Fast & Accurate

Our robust, ready-to-use assays, together with a high-quality oligo synthesis process ensures our customers a maximal PCR success rate (>97%). 

This, combined with a delivery time of 2-3 business days, the uniform PCR conditions of our assays and the avoidance of laborious normalization procedures, allows rapid variant validation resulting in the shortest turnaround times.

A variant identified on Monday has it's Sanger/NGS results on Friday ! 

Gene Coverage

Our product catalog includes 2 assay databases totaling up to almost one million assays, both covering almost 99% of the human protein coding exome: 

  • Database 1 encompassing over 285,000 assays with amplicon lengths ranging from 350 to 750 bp, is intended for high-quality DNA samples.
     
  • Database 2 is focused on degraded DNA samples - such as formalin fixed parafin embedded (FFPE) samples - and contains over 530,000 assays having lengths between 125 and 275 bp. Both databases were generated using our state-of-the-art primer design pipeline capable of generating robust and high-quality assays.

Prior to assay design, the exons of the canonical transcript for each gene were selected as target. Each target region was further extended at its 3' and 5' ends with a 25 bp spacer region to ensure sufficient sequencing quality at the start of the exonic target region (Figure 1). 

This is mostly of interest for Sanger sequencing, the quality at the beginning of a read is known to be sub-optimal. Resulting regions were then fed into our primer design pipeline.



Since our pipeline takes into account several quality parameters such as specificity and SNP/secondary structure content in primer annealing sites, generating assays using only the most stringent design parameters would result in ill-covered targets and remaining gaps. In this context, expecially the ever increasing number of SNP scattered across the exome is making it difficult to locate suitable primer annealing sites. 

To prevent this, our pipeline is equiped with an optimized relaxation cascade, which enables step-wise relaxation of design parameters in a controled manner. This workflow, combining design parameter relaxation with stringent in silico quality evaluations, allows us to generate high-quality assays while ensuring a maximal target coverage using a minimal number of assays (Figure 1).

More details on the primer design tool and associated relaxation cascade can be found in our "High-throughput PCR assay design for targeted resequencing using primerXL" paper (BMC Bioinformatics, 2017  ).

Frequently asked questions

Here are some common questions about our product.

While we did our very best to design perfect PCR assays, we are sometimes confronted with the constraints of the human genome sequence. As such, we occasionally needed to make a compromise (i.e. allowing a SNP in the primer annealing region or acknowledging possible off-target amplification):

  • 73.6% of our assays are labeled as perfect  meaning that we predict very high or ultimate specificity (because of the presence of at least 3 mismatches in each primer relative to a potential off-target sequence) and the complete absence of SNPs with a minor allele frequency higher than 1%. 
  • 10.9% of the assays are labeled as good  meaning that we predict good specificity (at least 2 mismatches per primer) and a maximum of 1 SNP. 
  • 15.5% of the assays are labeled as fine . In this category, we had to make compromises. Each assay comes with detailed information on predicted specificity and presence of possible SNPs. There is a very good correspondence between the predicted assay quality and the sequencing results (see product brochure, our peer-reviewed paper (Coppieters et al., 2016) or here). 
  • Assays marked with orange stars  have been wet-lab validated, while assays labeled with blue stars  have only been in silico evaluated.

There is a very good correspondence between the predicted assay quality and the sequencing results (see product brochure). If the specificity info - reachable by clicking on the stars assocated with a certain assay - in the assay detail windows shows that there are hits with 2/3 mismatches, this means that 2 and 3 mismatches are present in the forward and reverse primer, respectively.

Yes, you can. 

Universal tails can be added in two ways: 

  • One way is by supplying forward and reverse universal tail sequences in you profile and selecting "Yes" in the "Automatically add universal tail sequences?" field. This will automatically add the corresponding universal tails when adding assays to your cart.
  •  In addition, you have the option to add custom tail sequences to (a selection of) your primers once assays have been added to your cart. To do this, go to your cart and first select the assays for which you wish to add tail sequences. Next, click on the 'add/delete tails' submenu item in the 'action'-button menu and enter your forward and/or reverse tail sequence in the dialog box. 

In the same way, you can remove tail sequences from your assays by selecting


the desired assays and entering no sequences in the dialog box. Adding tails comes at no extra cost.

Add the PCR assay(s) of interest, Rainbow Probes/Primer or the Human Sample ID Kit to your cart and checkout. We accept bank/wire transfer payments and credit cards (via PayPal). All payments are in EUR currency. You can also purchase credits, and use the credit for future orders.

Our assays are carefully designed to have uniform primer annealing temperatures and amplicon lengths, to facilitate multiplexing. We have successfully multiplexed several assay panels for simultaneous equimolar amplification of DNA targets. 

For multiplex use, we recommend a dedicated multiplex PCR mastermix and reduced primer concentrations (20 nM final concentration for a given assay is typically a good starting point; to achieve uniform end-point amplicon concentrations, assay specific optimization of primer concentrations may be required.

Yes, we do. Just enter your target(s) of interest and desired amplicon size on the Custom assays page. If you click on the Add to cart button, the design will immediately be initiated upon checkout. Once completed, the assay(s) will be synthesized and delivered to you.

You can also request a quote first - e.g. for larger sets of targets - by clicking on the Request quote button or by using our Contact form. If additional information is required regarding your custom design request, we will contact you.

Our assays are carefully designed to have uniform primer annealing temperatures and amplicon lengths, to facilitate multiplexing. We have successfully multiplexed several assay panels for simultaneous equimolar amplification of DNA targets. 

For multiplex use, we recommend a dedicated multiplex PCR mastermix and reduced primer concentrations (20 nM final concentration for a given assay is typically a good starting point; to achieve uniform end-point amplicon concentrations, assay specific optimization of primer concentrations may be required.

Yes, we do. Just enter your target(s) of interest and desired amplicon size on the Custom assays page. If you click on the Add to cart button, the design will immediately be initiated upon checkout. Once completed, the assay(s) will be synthesized and delivered to you.

 You can also request a quote first - e.g. for larger sets of targets - by clicking on the Request quote button or by using our Contact form. If additional information is required regarding your custom design request, we will contact you.

We have done our utmost best to design primers that adhere to strict in silico quality controls ensuring robust and specific amplification of the target region of interest. Specifically, we avoid single nucleotide polymorphisms (SNPs) in the primer annealing regions (known to hamper efficient annealing or create allelic dropout), avoid secondary DNA structures (known to reduce PCR efficiency), and use a very stringent specificity prediction tool. 

Together, this ensures that the pxlence assays will amplify target DNA with very high uniformity and specificity. Several thousands of assays have been tested in the pxlence laboratory or in the lab of happy customers. Approximately 95% of the assays generate less than 2% off-target reads. More information is available in our product brochure or here.

The pxlence assays either come as lyophilized (100, 500 or 1000 reactions) or resuspended in buffer (100, 500 or 1000 reactions). For convenient use, forward and reverse primer come premixed, but can also be ordered separately. Primers are available in tubes or 96-well deep-well V-bottom plates. 

There is no price difference between tubes and plates or between lyophilized and resuspended in buffer. Please see our amplification guidelines for use of pxlence assays to amplify target of interest.

No, this is proprietary information, but you do get the amplicon context sequence as defined in Bustin et al., Clinical Chemistry, 2013 (Primer Sequence Disclosure: A Clarification of the MIQE Guidelines ), this is the true amplicon sequence +/- 20 nucleotides. 

Further, we provide detailed in silico quality control parameters for all assays (presence of SNPs and specificity score).

We recommend the Kapa2G Robust PCR kits for highly successful amplification of the target region. Several thousands of assays have been tested internally or by some of our early-access customers with very high success rate. We use the HotStart ReadyMix (kit code KK5701 or -02). 

pxlence assays should be used at 1x final concentration, and work with one universal PCR cycling program. Please also see our amplification guidelines.

Based on our survey with almost 200 respondents from all over the world, 70% indicated the need to confirm their exome, whole genome or targeted resequencing studies; 56% of these use Sanger sequencing, 19% massively parallel targeted resequencing and 20% both Sanger and massively parallel sequencing.

Pxlence assays and kits are sold for research use only. The data interpretation and clinical outcome remains the responsibility of the customer. Several of our customers use our assays and products in an accredited or quality controlled laboratory, upon internal validation. 

Of note, there may be other licenses required to use PCR in a commercial environment. It is up to the customer to ensure the required licenses.

Resources

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