Genotyping

Don't waste your time with genotyping !

 

Genotyping is often considered as a straightforward and easy step; in reality providing robust, accurate, and fast results is frequently more challenging that it seems.

 

 

Genotyping assay on complexe alleles or multiple regions of a locus	Simplified genotyping with one qPCR on specific region	Blastocyst genotyping
For more in depth validation of a mutant model	For minimal cost to the customer for established and verified line	Sperm genotyping

 

Genotyping services' flyer

For genotyping request please contact us

Customized genotyping services

We offer customized genotyping services for any mutation including knock-out, knock-in, transgenic or any other genetically modified rodent model. It is particularly useful during model creation for more in depth validation of a mutant model (QC of the mouse and rat lines). Many controls are necessary to validate complex alleles (e.g. after Flp or Cre recombination) or multiple regions of a locus.

Our service cover any genotyping step:

• Assay design and validation for new strategies or optimization of pre-existing strategies
• Genomic DNA extraction: different sample types can be processed, including: tails biopsies, ear tags, embryonic yolk sac and even non-invasive biopsies (hair, feces, buccal swab…)
• Samples treatments with one out of 6 methods available in routine (Universal or specific PCR and/or qPCR reactions, Target gene sequencing, Quantification with droplet digital PCR reactions… )
• Delivery of interpreted genotyping results (not only raw data).
• Delivery detailed genotyping protocol to all our users on request.

 

The service is available for all the scientific community; for any genotyping request please contact us

 

High-throughput genotyping

The fully automated service allows a high-throughput process of your customized protocol (and up to 40 universal assays in stock). We can process more than 200 000 assays (PCR, qPCR, ddPCR,..) per year.

We can also design a simplified genotyping with one qPCR on specific region for established & verified line:

• Allow only to distinguish WT / Heterozygote (Het) / Homozygote (Hom) for one specific allele
• Minimal cost for cohort establishment

Cryopreserved line genotyping

In our experience, data analysis over five consecutive years has revealed about 6% of inconclusive genotypes including not only genotyping errors but also animal misidentifications. This is consistent with the finding of Loyd that 15% of lines deposited to public repositories, do not carry the mutation specified by the depositor (Lloyd et al., 2015). At PHENOMIN-ICS, we propose a genotyping quality control of your mouse cryopreserved line with ethical and economical approach (3Rs):

Blastocyst genotyping: Validation of cryopreserved line by PCR on 10 blastocysts

• More than 400 cryopreserved lines validated to date
• Genotype line validation without the use of animals

Sperm genotyping: Validation by qPCR on frozen sperm

• Replace the use of animals
• Gain in time
• Secure project as rodent sperm is frozen

Technics

All used technologies are driven by in-house databases insuring integrated data management. We have six methods available in routine:

PCR: allele detection

• Infer the presence or absence of a mutated allele  (with appropriate controls)
• Vizualise the size of amplified DNA

qPCR: quantification Homozygote/Heterozygote

• Allows the determination of initial amount of a particular DNA sequence.
• Evaluate the presence of one or two copies of the allele

Digital droplet PCR: precise copy number

• Very precise quantification: sample division and counting of positive droplets over total droplets (+/- 5% over +/- 30% for qPCR) allow detection of low level differences.
• Evaluate the number of copies of a modified allele for Transgenic mice, CRISR/Cas9 founders for example

High Resolution Melting (HRM): mutations scanning

• Identify variations in a particular DNA sequence
• Detection of heteroduplexes due to the presence of a mutation

PCR + restriction fragment length polymorphism : mutations scanning

• Detection of a new restriction site due to the presence of a mutation

PCR + sequencing : mutations confirmation

• Sequencing of the mutated allele

PHENOMIN expertise

• Personalized genotyping service to fit our customer needs
• Delivery of interpreted genotyping results (not only raw data)
• Mendelian distribution / warning on inconsistent results if we received the parental genotypes
• All our models are delivered with their detailed genotyping protocol.
• Genotyping design for “archaeology” mouse model (from the original publications to design the mutant map, sequencing …)
• Continuous R&D activity to improve our technics and success rate and propose our customers the best approach for their projects.

1. Implication in international consortia (near 20 different consortia) to benefit from the last technics and scientific expertise.
2. We published on a new technical approach involved in the setting up of an alternative to reduce the number of animals used and improve the quality control to determine the ability of a mutant mouse line to recover after cryopreservation.
3. Celphedia Genotyping Work package (WP2.3) : Non-invasive genotyping of transgenic rodents (Anticipating the risks of legislative changes in Europe)

• Technical supports and advices: PHENOMIN-ICS went back to PCR genotyping basics to provide recommendations for reliable, rapid, cost effective, robust and adaptable to high-throughput genotyping protocol for any type of mutation.

PHENOMIN experts webinars

• WEBINAR SERIES 2020-2021, in partnership with Charles River
  • Mouse Study Reproducibility: The Impact of Genetic Background and Genotyping -  Replay is available online

PHENOMIN experts publications

• S Jacquot et al., 2019, Optimizing PCR for Mouse Genotyping: Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High-Throughput Genotyping Protocol for Any Type of Mutation.
• F Scavizzi et al., 2015, Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach.

FAQs

What can you do if the genotyping protocol sent with the mouse line doesn't work in your lab?

Troubleshooting genotyping assays can be challenging as multiple parameters from reagents to thermal cycling may be different in your labs in comparison with the provided protocol. In the publication Jacquot et al., we try to provide recommendations to design a reliable protocol and present critical parameters for optimization.

Another possible explanation is that the genotyped mice are not carrying the mutation or transgene as expected. Lloyd et al. found that 15% of lines deposited to public repositories, do not carry the mutation specified by the depositor. Careful validation of any new mutant line is highly recommended.

 

How can I validate the genotype of a cryopreserved line?

Cryopreservation of your mouse lines is an essential backup to secure your research project. In Scavizzi et al., we provide a blastocyst genotyping method for more ethical, simple and efficient quality control.

 

How long can I store my extracted DNA?

The right answer depends on your purification method. While kit or phenol/chloroform purified DNA can be preserved for a very long time, crude lysate usually deteriorate within a few weeks. Purified DNA can be stored for months at 4°C and for years at -20°C. Crude lysates can be stored at 4°C for up to one month with the DNA lysis described in Jacquot et al. For long term storage, we observed that these crude lysate can be safely frozen at -20°C.