KASP Genotyping Services FAQs

If you are new to LGC and would like to run a genotyping project in our service laboratory, please follow these steps:

1. Obtain a quotation from your sales rep for your project. If you need to be put in contact with the appropriate sales rep for your region, please contact us.
2. Tell us which SNPs you are interested in by completing a short SNP submission form and DNA sample submission form for your genotyping project.
3. Submit a purchase order, and your completed SNP submission and DNA sample submission forms to genomics@lgcgroup.com. Please ensure that you include your customer number and quotation number in the e-mail.
4. You will be assigned a project number and a project manager for your genotyping project. Your project manager will liaise with you throughout your project, from submission of your DNA samples through to project completion.

Turnaround times measure from the receipt of your samples to the delivery of your data. The standard turnaround time for genotyping is 4-8 weeks. A rapid turnaround time of 2-3 weeks is possible, subject to an additional charge.

LGC offer a number of SNP assays that can be used to detect whether individual animals are resistant or susceptible to Scrapie. Scrapie is a fatal disease that affects the nervous system of sheep and goats. The disease causes affected animals to scrape off their fleeces against trees or rocks, hence the name Scrapie. Scrapie is infectious and therefore the only way to contain it is to quarantine and destroy affected animals. In the UK, the Government has put in place a National Scrapie Plan that encourages breeding from sheep that are naturally resistant to the disease. Individuals that are identified, through genotyping, as resistant can be used in the breeding of future generations.

If you do not have sufficient genomic DNA for the number of SNPs that you wish to run, it is possible to perform Whole Genome Amplification (WGA). WGA is a PCR technique that is used to produce large quantities of DNA from a small amount of starting material. There are a number of methods for WGA, and LGC favours use of the primer extension pre-amplification (PEP) technique. PEP employs the use of randomly synthesised 15-mer oligonucleotides, referred to as polyN15, that bind at sites throughout the genome and act as primers to enable DNA replication. LGC uses our in-house enzyme, Klear Taq, and buffer system to perform WGA reactions.

To perform WGA on your samples, LGC requires a minimum of 50 ng of genomic DNA per sample. If the quality of the starting material is good, the product is typically amplified to a concentration of 500-1000 times higher than that of the starting material.

Although LGC can offer DNA quantification services we do not typically perform quantification of DNA after Whole Genome Amplification. Prior to running a full-scale genotyping experiment on WGA samples, we run a small-scale genotyping test to determine the optimal DNA dilution for such samples. If you require quantification of your WGA DNA samples by UV measurement, we can offer this but there will be a charge.

All samples that are sent to LGC are received by our sample receipt team and tracked in our LIMS system. When your samples arrive, the team will ascertain that the samples are all in good order and ready for cold storage. If required, we will barcode your samples prior to storage. Your genotyping project will have been assigned to a project manager, and they will be responsible for coordinating all work on your samples. The appropriate dilution plates will be created from your samples, and these will be stamped into either 384-well or 1536-well reaction plates and dried down in drying ovens. The stamped plates then move to the dispensing laboratory, where the relevant SNP Assays are run over your samples using our Meridian dispensing robots. Reaction plates are laser sealed and subsequently undergo the KASP thermal cycle in one of our Hydrocyclers. At the end of the thermal cycle, the fluorescent signal is read on a BMG PHERAStar plate reader and imported into our LIMS database. You project manager will then be responsible for performing detailed analysis of your data before the results are sent to you.

We only accept DNA for genotyping in our laboratory. If your samples are RNA, please perform cDNA synthesis and subsequent quality checks before submitting them for genotyping.

For a genotyping project, there is no minimum or maximum number of SNP assays. You can just run one SNP assay across all your samples if this is all that you require.

As KASP genotyping is not a quantitative assay, it is not essential to perform an RNase treatment on your samples.

Aliquots of assays from completed genotyping projects are stored onsite indefinitely. If you have additional samples that you wish to have genotyped with these existing assays, and there is stock remaining of the original aliquot, you will not be charged for a new assay aliquot and will only be charged for the genotyping service. If there is insufficient assay left to genotype all of your additional samples, you will be charged an assay re-stock fee.

Following completion of the initial 35 cycles of PCR, all genotyping reaction plates run at LGC are read on a BMG PHERAStar plate reader. This initial read data is visually inspected by a member of the genotyping team to assess the progression of the PCR reaction. The plates are then recycled (3 cycles per recycle step) and read after each recycle step. The laboratory operator visually inspects the read data after each recycle step and, once they are satisfied that the PCR reaction has reached endpoint, indentifies plates as completed. At this stage, our in-house Kraken™ software will automatically call genotypes for your samples. Your project manager will access the plate read data in Kraken and perform a detailed analysis of the data. This may require them to correct the automatically called genotypes that Kraken has given. Version one of your genotyping results is then exported within the Kraken system. A second project manager will then second check these results and verify or change them in collaboration with your project manager. The results are then ready to send to you, the customer.

Once your genotyping project has been completed, your project manager will send you a results file in an Excel .csv format. You can open this type of file in Excel and work with the data in there or import the file into our SNPviewer software to view the data graphically. In SNPviewer you will be able to see the clustering of your datapoints. SNPviewer (free-of-charge software) can be downloaded from our website.

LGC do not offer bioinformatics services for genotyping projects.

SNPviewer produces a Cartesian plot of the FAM and HEX data, using values that have been normalised using ROX passive reference dye. The scale represents fluorescence intensity units from our BMG PHERAstar plate readers. These intensity values are arbitrary, with FAM values plotted on the X axis and HEX values plotted on the Y axis. Due to the differences in FAM and HEX excitation and emission peak heights, there is a slight skew towards FAM that is reflected in the minor differences in scale values for each axis.

If your DNA is extracted in our service laboratories, quantification by spectrophotometry will only be performed if specifically requested within your project (this will incur an additional charge). DNA extracted in our service laboratories can be transferred directly to the genotyping laboratory for a genotyping project, and dilution tests will be performed to determine the optimal dilution for your samples.

If you perform DNA extraction yourself and send the DNA samples in to our genotyping laboratory, your project manager will request information regarding the concentration of the samples and the method used for quantification from you. LGC do not quantify customer DNA samples prior to genotyping, although this can be offered as a service if required.

For more information, please visit this page. This document also provides more information about DNA quantification methods.

Yes, providing the starting material is good, the resultant WGA material works well with the majority of SNP assays. In theory, there will be some bias towards and against some areas of the genome, but this rarely causes major issues. LGC offers WGA (PEP-based) as a service. This WGA method results in fragment lengths of 500-600bp that are more than long enough to perform KASP genotyping on (max amplicon length for KASP is approximately 100 bp). Samples that have been amplified using MDA WGA will work equally well with KASP genotyping.

To normalise DNA samples in-house, LGC bring an aliquot of the neat DNA to a desired concentration (typically 50ng / µL) in a 96-well plate. Note that the standard maximum volume per normalised sample is 1 mL. Any remaining neat DNA can be shipped back to the customer where requested.

• Whole blood
• Buffy coats *
• Buccal swabs
• Saliva OraGene tubes
• Saliva (Other)
• Serum *
• Human tissue **
• Animal tissue punctures **
• Cells / Cell cultures (please provide number of cells in case of RNA extraction)
• Plant leaves **
• Plant seeds **
• Plant roots **
• Other plant material
• Fin clips (on ethanol)
• Faecal matter
• Urine
• Paper blood spots
• Many other sample types - please contact our technical support team

* Serum and buffy coat yields can vary greatly dependent on individual samples; we advise that a pilot study is performed for a reliable prediction about the DNA yield for your specific samples.

** Please supply in 96-well plates in a sorted manner.

LGC can extract DNA from samples of any volume up to 50 mL. For sample volumes greater than 50 mL, customised options are available. Please contact our customer service team for more information.

• Concentration normalisation of DNA extracts required (normalised DNA will be prepared in 96-well plates.)
• Quantification of DNA / RNA concentration (UV measurement)
• Cherry picking of samples (Most suitable from 96-well plates, or 384-well plates in some cases. Cherry picking from 1536-well plates is not feasible. Please ask your project manager or our scientific support team for more details)
• Sample transfers
• Sample management
• Sample tracking
• Plating of samples
• Plate transfer
• Additional aliquots of DNA
• Sample storage for KASP genotyping (free of charge)
• Quality check of RNA with Agilent 2100 Bioanalyzer
• Gel electrophoresis analysis
• Genotyping QC (3 KASP assays incl. gender test)
• Whole Genome Amplification (WGA)
• Genotyping projects
• Sequencing projects

Please contact us for more information.

We would expect to obtain around 20-30 µg of DNA per 1 mL of human whole blood from a healthy individual. The DNA yield will be dependent on several factors such as the health of the individual, the technique used to obtain the sample, and the way that the sample has been stored.