Genotyping laboratory workflow
Once your KASP assays have been prepared and your DNA samples are received, laboratory work will commence for your genotyping project.
Upon receipt of your DNA samples, LGC will commence laboratory work for your genotyping project.
The key stages in the laboratory workflow are:
- Sample plate preparation - LGC prepare sample plates for genotyping using either 384-well or 1536-well format plates depending on the number of SNPs / indels and samples being genotyped.
- Assays run on sample plates – assays will be dispensed into the genotyping sample plates. The plates are sealed, thermally cycled, and then read to obtain fluorescent data.
- Genotyping cluster data scored - the fluorescence data read from the sample plates is plotted and genotypes are assigned to samples based on clusters.
Sample plate preparation
DNA samples provided by the customer are stamped into reaction plates in preparation for KASP genotyping reactions. These can be either 384-well or 1536-well format plates depending upon the scale of the project. DNA samples are stamped using our repliKator™ instrument. All reaction plates are barcoded and tracked to your project within our Kraken™ database. DNA samples are dried down into reaction plates to ensure stability of samples during storage.
Assays run on sample plates
Once the KASP assays within your genotyping project have been assembled and validated (where applicable), they can be dispensed across the DNA sample plates. Dispensing is performed using our Meridian2 ™ dispensing robot. Plates are laser-sealed using the Fusion™ and are thermally cycled in one of our
Hydrocycler2 ™ instruments. 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.
Genotyping cluster data scored
Once the laboratory operator is satisfied that the PCR reaction has reached endpoint, the reaction plate will be identified 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.