Frequently Asked Questions
When performing the recommended QC steps, what should my PCR products look like?
When running the PCR products on a 2 % agarose gel, successful amplification can be detected as a clearly visible but dispersed band, whereas the negative control should not display any amplification product for the run to be valid. Note that when using FFPE-derived DNA, the amplification will be hardly visible making this QC step redundant for FFPE-derived DNA.
In Figure 1, we show an example for the 2 plexes of the CFTR MASTR Dx.
Alternatively, a microfluidic-based method can be used (examples are Agilent Technologies 2100 Bioanalyzer and 2200 TapeStation, Perkin Elmer’s LabChip® GX).
In Figure 2, the PCR amplification of the Universal PCR performed on the 2 plexes of the CFTR MASTR Dx is shown using LabChip GX.
The expected amplicon sizes are:
- Multiplex PCR: the fragment size ranges between 250 and 450 bp for germline MASTRs, and between 150 and 300 for somatic MASTRs
- Universal PCR, MID and adaptors are added, increasing each amplicon size:
- MID for Illumina MiSeq: + 97 bp
- MID for Ion PGM System: + 84 bp
- 454 MID: + 70 bp
- Short Read Amplification: + 0 bp
Figure 1. QC step on 2% agarose gel for both the first and second PCR (both plexes of a CFTR MASTR Dx on sample and negative control)
Figure 2. QC step on LABchip GX for the first PCR (both plexes of a CFTR MASTR Dx on 4 samples and H2O control).
For the Universal PCR, we recommend to perform a fragment analysis on the FAM-labeled amplicons (according to our Instructions in the appropriate IFU Part II) using an ABI capillary sequencer with GeneScan module. The expected chromatogram per plex can be found on the MASTR-specific GS Reference Pattern, which can be downloaded from our website when choosing the desired MASTR under the Products section.
What is the recommended minimal coverage?
Multiplicom recommends the following minimal coverage per allele:
|Minimal coverage||Germline MASTRs||Somatic MASTRs|
|SNV (per allele)||CNV (per amplicon)||SNV (per allele)||CNA (per amplicon)|
|Illumina NGS Systems||20||200||20||NA|
|Ion PGM System||50||300||50||NA|
SNV = single nucleotide variant
CNV = copy number variation
CNA = copy number aberration
SRA = Short Read Amplification kit
NA = not applicable
What are the critical steps of the workflow?
DNA quality and integrity: DNA quality needs to be good for optimal amplification.
- DNA integrity: Degraded DNA will result in favored amplification of short amplicons and gradual loss of longer amplicons.
- EDTA content of the buffer in which the DNA sample is diluted: EDTA has an inhibitory effect on the PCR amplification; it is recommended to keep the EDTA concentration below 1 mM, TE buffer. DNA derived from FFPE material already contains inhibitory components, therefore we recommend keeping the EDTA concentration below 0.1 mM (TE-4 buffer) for somatic MASTRs.
Preparation of the used DNA: prepare the appropriate dilution just prior to use. Many steps in this process need vortexing, followed by spinning down the sample at 12,000 x g for 10 s.
- MASTR: Sufficiently dilute the DNA sample enabling pipetting of larger volumes: e.g. take 5 µl of a 10 ng/µl sample instead of 1 µl of a 50 ng/µl sample. To make the required dilution, use molecular biology water or TE-4 (don’t use regular TE, as this contains 10x more EDTA) and vortex well.
- MID: Dilution of the Multiplex PCR (used as template) is essential. This dilution needs to be executed in a 2-step process when exceeding a dilution factor of 100x (e.g. 2 µl on 100 µl followed by 2 µl on 40 µl, for a 1/1000 dilution). Correct vortexing and spinning between the different dilution steps is critical.
Preparation of the PCR mixes (for both MASTR and MID PCR). Important aspects are:
- Thaw all needed reagents completely, and vortex the vials each time prior to use(to ensure the use of the optimized buffer composition).
- To avoid pipetting too small volumes of the viscous Taq DNA polymerase (less than 0.5 µl, e.g. less than 7 samples for the MASTR reaction mixes): dilute the Taq 1/10 in molecular biology grade water, and make sure to vortex this dilution properly before use. Make this dilution freshly, do not store.
- After adding the appropriate volumes of all the reagents to a clean vial (master reaction mix), the resulting mix needs to be vortexed and spun down thoroughly before dividing over the PCR tubes or plate.
- When the DNA is added to the master reaction mix, vortex and spin down the PCR tubes or plate thoroughly before placing it in the PCR cycler.
The ramp rate of the PCR machine needs to be between 1 and 2°C/s for all heating and cooling steps. If the ramp rate is too high, the largest amplicons will not be amplified efficiently.
After the MASTR PCR, immediately continue with the Universal PCR step. Otherwise, store amplified products between ‐15°C and ‐25°C. Do not store the amplified products for more than 1 h at 4°C.
When performing the purification of the mixed amplicon libraries:
- Use the correct volumes for amplicon library and Agencourt AMPure XP beads. An incorrect ratio amplicon library/Agencourt AMPure XP beads can result in the loss of the shortest amplicons during purification.
- Perform the correct number of purification rounds.
Both factors are indicated in section 3 of the Assay Specification Sheet.
Why are there different amplification reagents (AR) for different MASTRS? And why is this AR buffer supplied in the MASTR kit, while it is needed with the MID kit?
The AR buffers are used to optimize the performance of the MASTR. Since every MASTR targets different genomic regions, it requires a careful optimization. Multiplicom has currently three different Amplification Reagents (AR1-3), with specific content, enhancing amplification of the targeted genomic regions.
For the MASTR kit (Multiplex PCR) the enhancing component is already added to the MASTR-specific PCR Mixes. However, since our Complementary MASTR Products (MID and SRA kits) can be used for any MASTR, the required enhancing component needs to be added for Universal PCR, and will be supplied together with the MASTR.
- Libraries from a MASTR using AR3 as amplification reagent for the Universal PCR can NOT be measured using a fluorometric method, such as Qubit® from Life Technologies.
- Note that the same AR buffer from different MASTRs (eg, AR for BRCA MASTR or CFTR MASTR) has exactly the same content, even though the vial is labeled MASTR-specifically.
What is a key-code and how to use it?
- gives access to the complete and most updated set of downloads
- has to be entered on our website (http://www.multiplicom.com/keycode)
- can be found printed on the box label of your kit, and on the kit insert
Each key-code correlates to:
- a specific MASTR
- a specific version of that MASTR
- a specific set of downloads for that MASTR
- a validation round with specific protocol, claims and disclaimers (for CE-IVD labeled MASTR Dx assays)
… but does not correlate to:
- a specific batch (production LOT) of the MASTR
It is highly recommended to log the key-codes on the kits and compare each time before using a new kit.
I suspect something went wrong with the amplification steps of the protocol, but I do not know where exactly and why. Can you help me?
To get a clear idea of what is going on, we would strongly recommend some intermediate QC-steps as described in the IFUs:
- After the first, multiplex PCR: run 5 µl of the amplified PCR product on 2% agarose gel, or use a microfluidic-based method (eg, Agilent Technologies 2100 Bioanalyzer and 2200 TapeStation, Perkin Elmer’s LabChip® GX).
- After the second, Universal PCR: also agarose or microfluidic-based methods can be used, however, Multiplicom prefers the review (using our MAQ-S) of fragment analysis data generated by an ABI capillary sequencer (with GeneScan module) on GS-labeled PCR product.
With this information you can contact your local distributor or your sales representative. Online form can be found at http://www.multiplicom.com/contact, at which also QC files can be uploaded (fi, agarose images and .fsa files). Please make sure to describe the encountered problem clearly, and indicate what is to be seen on the different images and files.
Which software packages for data analysis does Multiplicom support?
We support the use of SeqNext (JSI) and Sophia DDM (Data Driven Medicine, Sophia Genetics). These software packages are also used for our CE-IVD products. For SeqNext we offer a Definition file (.zip file containing either the .tsv file and .fas files, or a SeqNext-specific .bed file for defining the reference sequences).
Currently we don’t offer support on other software packages (eg, Workbench by CLC Bio, NextGENe by Softgenetics, Genious by Biomatters, SeqMan NGen by DNAStar, …), but we do offer the required info on the position of our trimmed amplicons (BED-file) as well as the primer sequences. These files are available as downloads when entering the key-code of your MASTR kit at http://www.multiplicom.com/keycode.
What causes the additional peak in my genescan pattern?
In case the additional peak is very small (100-160 bp; outside the range of the amplicon sizes), most likely primer dimer formation has occurred.
In case it is an extra peak in the expected range of the amplicon sizes, this peak might suggest the presence of an indel in one (or both) allele(s) covered by the affected amplicon. These indels typically combine the appearance of an extra peak with a decrease in peak area of the original peak (heterozygous indel: peak areas of both peaks are comparable), or the appearance of an extra peak with a total loss of the original one (homozygous: peak area of the new peak resembles the expected peak height for the original amplicon). The extra peak can occur at smaller (deletion) or larger (insertion) amplicon size compared to the original amplicon.
In case of an unclear discrepancy between the fragment analysis data and the sequencing data, please contact customer service: email@example.com.
Which negative control samples do I have to include for the different parts of the workflow?
For both the Multiplex PCR of the MASTR kit and the Universal PCR of the MID/SRA kit, we recommend to include a negative control sample:
- For the MASTR: water
- For the MID/SRA: continue with the water control used in the MASTR
- Use the same master reaction mix as prepared for the actual samples.
- Perform every step of the protocol with the negative control sample, similar as the patient sample.
- Run the amplification along with the actual samples in the PCR cycler.
- Review the negative control when performing the QC steps described in the IFUs (when using agarose, try to run the negative control on the same gel as the other samples).
How should I treat the different vials from the Multiplicom kits before use?
- Each time you use a vial (except Taq DNA polymerase), remove from the freezer and allow complete thawing on ice.
- Taq DNA polymerase should be taken from the freezer just prior to use.
- Vortex the vials (except Taq DNA polymerase) thoroughly.
- Centrifuge all vials (including Taq DNA polymerase) at 12,000 x g for 10 s to prevent loss of reagent/enzyme when opening the vial.
- For Taq, due to the viscosity, take extra care when pipetting and avoid droplets at the exterior of your pipet tip.
- Place the vials back in the freezer as soon as possible, and keep the number of thaw-freeze cycles below 5 (preferentially ≤ 3, if desired aliquots can be made and stored as such).
Note that for each of the reagents/enzyme an excess for a few reactions is provided. By carefully following the procedure above (as also mentioned in the IFUs), a shortage can be avoided.
Is a certain SNP covered by the library of Multiplicom?
This can be checked using the information provided in the BED-file (.txt document) representing the trimmed amplicons:
- Open the BED-file in NotePad, select all and copy
- Go to UCSC Genome Browser (genome.ucsc.edu)
- Select the tab "My Data" → "Custom Tracks”
- Make sure assembly "Febr. 2009 Human (GRCh37/hg19)” is selected
- Paste the copied cells at "Paste URLs or data" → "Submit"
- Select “Go to Genome Browser”
- Enter the SNP of interest (rs number)
- Is the SNP covered by the User Track bar: yes or no?
Why do some amplicons include short intronic regions (< 10-15 BP)?
In general, Multiplicom strives for the inclusion of a minimum of 30 bp intronic region at every intron-exon boundary. However, to guarantee correct annealing of the designed primers we avoid repetitive regions and SNPs. As a result, some primers are located closer to the intron-exon boundary than 30 bp.
Remark: The intronic region is standard shorter for Somatic MASTR (Plus) assays. The minimum included intronic region varies between 1 to 5 bp depending on the assay at hand.
How can I perform an accurate quality assessment of my FFPE-derived DNA?
DNA derived from FFPE tissue can be a cumbersome source of input DNA for downstream PCR-based processing. Several factors contribute to this suboptimal performance of FFPE-derived DNA:
- formalin fixation can lead to DNA crosslinking and DNA degradation;
- presence of PCR inhibiting factors and
- low DNA concentration.
Measuring FFPE-derived DNA using spectrophotometric and/or fluorometric methods provides information on the amount of DNA present but not on DNA integrity and the presence of PCR inhibiting contaminants in the DNA solution. Since often limited amounts of FFPE-derived DNA is available, it is not desirable to perform several consecutive quality measures.
Multiplicom’s QC Plex assay offers a one-step all-in-one solution. The resulting QC Plex DNA quality coefficient (DQC) value determines the volume of input DNA required for successful amplification of FFPE-derived DNA (see table below).
|DQC-value||DNA quality||Volume input DNA per plex||Molecular biology grade water|
|> 5||Excellent DNA quality||1 µl||4 µl|
|5.0-1.1||Good DNA quality||2 µl||3 µl|
|1.0-0.2||Acceptable DNA quality||5 µl||-|
|< 0.2||Poor DNA quality||It is advised not to use the sample in a MASTR assay|
How much DNA do I need for a MASTR?
The MASTR only needs a small amount of input DNA:
- For high-molecular weight DNA (derived from blood or FFT-derived DNA); only 20 ng per plex is required.
- For FFPE-derived DNA, the amount depends on the fragmentation due to formalin-fixation. The QC Plex gives you a measure of the degradation and an indication of the amount of DNA necessary per plex. Amounts can be as low as 4 ng/plex but to take into account tumor heterogeneity, we advise 20 ng/plex
What are the sequences of Multiplicom’s Universal Tags, MID and adaptors?
|MID for Illumina MiSeq||MID sequences||See IFU333|
|MID for Ion PGM System||MID sequences||See IFU243|
|454 MID||MID sequences||See IFU236|
* Universal Tags 1 and 2 are incorporated during the multiplex PCR (MASTR assays), and used as adapters during annealing of the primers for the Universal PCR (MID kits for incorporation of MPS-specific Molecular IDentifier and adaptor sequences).
Are Multiplicom kits compatible with all MPS systems?
Yes, the Multiplicom MID kits make your library compatible with the MPS system of your choice. Specifically:
- MID for Illumina MiSeq: two different kits with 8 p7 and 6 p5 MID primers each.
- 454 MID kits: four different kits to incorporate the A & B adaptors and 8 MID primers each.
- MID for Ion PGM: two kits to incorporate the A & P1 adaptors (in both orientations) and 8 different MID sequences.
- For all other MPS instruments generating short reads (≤200bp, such as Life Technologies Ion PGM™ using their Ion PGM™ Sequencing 200 Kit v2) or for MPS instruments for which Multiplicom does not have an MID kit available yet (future technologies), we offer the Short Read Amplification kit.
Can I really perform copy number variation (CNV) analysis using my sequencing data?
Yes, for a selected set of MASTRs. Starting from high-molecular weight DNA, our MASTRs behave uniform enough so that read counts can be used as a measure for copy number. Normalizing the readcounts identifies genomic regions with more or less reads, meaning an amplification or deletion specifically. To simplify this process, use our CNV Calculator (http://www.multiplicom.com/calculators). If your favorite MASTR is not listed, please contact our customer service (firstname.lastname@example.org).
For CNV analysis, always keep these tips in mind:
- All samples should be amplified under exactly the same conditions (ie, same master reaction mix and same PCR run) during both the multiplex and the Universal PCR.
- Deeper sequencing is required for CNV analysis (on average 3x deeper compared to what is recommended for single nucleotide variant (SNV) analysis),
- When using a set of known samples as references (no CNVs), the libraries of these samples should be constructed together with the unknown samples,
- When using all samples of the run as references, only 40% of samples from the total set is allowed to have a CNV.
Why would I need an HP and/or MAQ kit?
These kits serve as an extra confirmation of either homopolymer variants (HP) or changes in copy number (MAQ).
HP assays complement the limitations of MPS platforms to identify variations in homopolymer regions. Specifically, emulsion-PCR-based sequencers (454 and Ion PGM Sequencing) have limitations to detect variation with any stretch >6bp and Illumina sequencers with stretches >10bp. HP assays will generate fragments of different size with different HP regions that can be analyzed on a capillary flowcell.
MAQ stands for Multiplex Amplicon Quantification, and serve as a confirmation for regions that are suspected as altered in copy number. The MAQ will analyze the amount of PCR products of the ROI and compare it with a control genomic location using a capillary flow cell and the MAQ-S software.
What does MASTR and MID stand for?
MASTR stands for Multiplex Amplification of Specific Targets for Resequencing. Multiplicom’s MASTR kits enable the specific PCR-based amplification of a given set of genomic Regions of Interest (ROI) in a limited number of multiplex PCR reactions or plexes. More information on the currently available MASTRs can be found under the Products section.
MID stands for Molecular Identifier (or barcode). Multiplicom’s MID kits incorporate molecular identifiers to link the library to a specific sample and to make your library compatible with the MPS of your choice.
More information on the currently available MID kits can be found in the product section.
Multiplicom is ISO 13485 certified. What does it mean?
The quality management system of Multiplicom has been assessed and certified, by a third party, as meeting the requirements of the International Organization for Standardization (ISO) 13485 standard. ISO 13485 certification ensures the consistent design, development, production and delivery of in-vitro diagnostic kits that are safe for their intended purpose.
Where can I obtain a Material Safety Data Sheet for Multiplicom’s products?
Multiplicom does not manufacture nor distribute products containing dangerous substances. Therefore, an MSDS is not required.
How long does it take to receive my order?
You will receive your kits within 6-10 days, if the ordered product is in stock.
What is the core technology of Multiplicom about?
Multiplicom has a variety of products in its portfolio: kits enabling library preparation for MPS (MASTR and MID), kits for accurately calling the presence of an indel in homopolymer stretched (HP), and kits enabling CNV analysis (MAQ). All these kits are based on highly multiplexed PCR.
Multiplicom’s multiplex PCR technology uses a proprietary software (MultiplexerTM) tool to design primers supporting the best possible multiplexing strategy in combination with solid expertise in multiplex PCR assay optimization. This results in the generation of highly efficient, low cost assays to establish a wide range of clinical and diagnostic applications.
For more information, please follow this link: http://www.multiplicom.com/technology.
What is MPS, and is it something else than NGS?
MPS (Massively Parallel Sequencing) is exactly the same as NGS (Next-Generation Sequencing).
Both refer to the second generation of sequencing.
What is the shelf life of Multiplicom’s MASTR and MID products?
Multiplicom products have a shelf life from 12 up to 24 months, counting from production date. Products are shipped with a minimal remaining shelf life of three months. Please check the precise expiry date, which can be found on the box label of your kit.
What is ‘soft-clipping’, and how can it affect variant calling?
A soft-clipped sequence is the part of a read that is not in alignment with the reference sequence (an unmatched fragment in a partially mapped read). This unaligned sequence is therefore removed from the analyzed data. Depending on the used data analysis solution, this is a phenomenon that might occur when a variant (nucleotide substitution or indel) is present in close proximity to the end or at the extreme end of the sequencing read. This is due to insufficient sequence context 3’ from the variant. A possible solution is to align the reads prior to primer trimming.
|BRCA MASTR and BRCA Tumor MASTR Plus (NM_007294.3) = BIC / IARC database||RefSeq (NG_005905.2)||BRCA Hereditary Cancer MASTR Plus (NM_007294.3 + NM_007300.3) = RefSeq|
|-||-||Exon 1 (NM_007300.3)|
|Exon 2 - 3|
|Exon 5||Exon 4b||Exon 4|
|Exon 6||Exon 5||Exon 5|
|Exon 7||Exon 6||Exon 6|
|Exon 8||Exon 7a||Exon 7|
|Exon 9||Exon 8||Exon 8|
|Exon 10||Exon 9||Exon 9|
|Exon 11||Exon 10b||Exon 10|
|Exon 12||Exon 11||Exon 11|
|Exon 13||Exon 12||Exon 12|
|-||-||Exon 13 (NM_007300.3)|
|Exon 14||Exon 14a||Exon 14|
|Exon 15 - 24|
How can I place an order for Multiplicom products?
Multiplicom is accepting:
- orders issued through the Customer Portal, which can be reached via the 'Place & Review Orders Tab' in the main menu of the website
- orders issued by e-mail message to email@example.com
- orders transmitted by fax to the number +32 3 400 23 1
- orders mailed by post to: Multiplicom NV, Galileïlaan 18, 2845 Niel, Belgium
Where do I access the instructions For Use (IFU) of a CE-IVD labeled kit?
- On the label of each of the boxes, you will find the key-code number
- Open the CE-IVD kit. Inside the box, you will find a small card with the key-code instructions to download the protocol
- Once you get the key-code numbers from each of the box go to the following URL to download the instructions: http://www.multiplicom.com/keycode
What is the delivery-time for Multiplicom products?
Shipments take place every Tuesday. Orders arriving before Friday will be shipped on the next Tuesday, if ordered products are on stock. The transport goes with express courier enabling delivery of most orders within the week of the shipment day.
What kind of equipment does Multiplicom recommend to ensure the best results?
- DNA-extraction: Qiagen's extraction kits, QiaCube & PSS (automatic DNA-extraction)
- PCR equipment: we work with the Verity from Applied Biosystems, older systems or different manufacturers can also be used
- Fragment Analysis: 3730XL from Applied Biosystems or other comparable capillary systems
What is the minimum quality standard for DNA, to be used in our assays?
- OD260/280 ratio ≥ 1.8
- Concentration ≥ 10 ng/µl in H2O
- No DNA degradation visible on agarose gel
How to store DNA?
- Long-term storage: -20 °C
- Short-term storage (max 1 day): 4°C
- Prevent freeze-thaw cycles
How should I treat the different vials from the Multiplicom kits before use?
- Each time you use a vial (except Taq DNA polymerase), remove from the freezer and allow complete thawing on ice
- Taq DNA polymerase should be taken from the freezer just prior to use
- Vortex the vials (except Taq DNA polymerase) thoroughly
- Centrifuge all vials (INCLUDING Taq DNA polymerase) at 12,000 x g for 10 s to prevent loss of reagent/enzyme when opening the vial
- For Taq, due to the viscosity, take extra care when pipetting and avoid droplets at the exterior of your pipet tip
Note: for each of the reagents/enzyme an excess for a few reactions is provided. By carefully following the procedure above (as also mentioned in the IFU's) a shortage can be avoided
What could cause that the percentage of on-target sequences is lower than expected (although all regions of interest are covered)?
The presence of many reads not aligning to the regions of interest defined by the specific MASTR used can result from a high percentage of primer dimers. The occurance of these primer dimers can have two reasons:
- the Multiplex PCR (MASTR assay) might be stored at 4°C for too long: longer than 1 hour (including the storage step after the PCT program in the thermo cycler)
- the amount of DNA input is too small (especially important for somatic MASTR's) resulting in an unfavorable primer/DNA ratio (too high)