Behind the Scenes at Basepaws
You’ve purchased a CatKit, collected some of your cat’s DNA and shipped it back to us. Exciting stuff! Wait, but what happens next? The time has come for us to unravel a little bit about our recipe to unfolding your feline’s DNA secrets. No, not everything - there is still a lot we don’t know ourselves, and every sample helps get us closer. But we want to tell you a bit about us, inside and out. Let’s take a quick look behind the curtains and see what really happens once your sample reaches the Basepaws Labs!
1. It all starts with a little bit of DNA
The entire Basepaws mission builds and revolves around DNA. We want to learn more about feline ancestry and evolution, their health, personalities and behavior, and we use DNA as our main source of information to do this. DNA, or deoxyribonucleic acid, is a complex organic molecule consisted from two polynucleotide chains. Polynucleotide chains consist of nucleotides – monomers built from three components: nitrogen-containing nucleobases (A, G, C, T), deoxyribose (sugar) and a phosphate group. The order of these nucleotides in the DNA determines the coding information for the synthesis of every gene product in the body. When we extract the DNA from your cat's cells, we must decode the information written within it. This allows us to decipher your cat's uniqueness.
2. DNA extraction
DNA is a very long molecule and in order fit inside the cell, it has to be packed very tightly with the help of proteins. For this reason, the extraction and isolation of the DNA molecule from the cell is a challenging job. Most commonly, cells used for DNA extraction in cats are cheek cells, hair follicle cells or blood. Here at Basepaws we employ the buccal swabbing technique for DNA sampling and we extract the DNA from your cat's cheek cells. Please follow this instruction video for help on DNA sample collection from your cat:
In order to extract the DNA from your cat's cheeck cells, our lab uses our own proprietary DNA extraction methods. We use chemicals and enzymes to break cellular components and then we use other reagents to isolate the DNA. It usually takes about 2 days for the extraction process to complete, and dozens of individual steps! The DNA has to be purified and cleared from all the other components in the sample. All the carbohydrates, lipids, proteins and other compounds found in the samples are removed with appropriate reagents until, in the final result, we get clean DNA we can work with. Pweh!
3. DNA sequencing
As mentioned above, DNA molecule is consisted of polynucleotide chains. To determine the precise order of these nucleotides in the DNA molecule, we have to sequence it. Different methods or technologies can be used to determine the order of the four bases (A, G, T and C) in the DNA sample. The basic methods for DNA sequencing are Maxam-Gilbert and Sanger sequencing methods. Today, however, more advanced, faster, cheaper and straightforward methods are being developed and used instead. These are the “High-throughput” methods (or “New Generation Sequencing”) and the “Third Generation Sequencing methods”.
1) The basic sequencing method: In the Sanger sequencing technique, the more broadly used of the two basic methods, the DNA is copied many times in a way in which the resulting fragments are terminated in different positions. This results in different lengths of the synthesized fragments. The terminating nucleotide of each fragment is marked with fluorescent “chain terminator”. These fragments are then run through a gel matrix in a process called capillary gel electrophoresis. The shortest fragments will pass through the gel fastest, followed with the longer sequences one by one. At the “finish line”, the terminating labeled nucleotide will be detected. This is how we can determine the order of the nucleotides in the target DNA.
“Although genomes are now typically sequenced using other methods that are faster and less expensive, Sanger sequencing is still in wide use for the sequencing of individual pieces of DNA, such as fragments used in DNA cloning or generated through polymerase chain reaction (PCR).” - Khan Academy.
2) High-throughput methods (NGS methods): Based on the basic methodology, researchers have eventually developed automated DNA sequencers which significantly fastened the entire sequencing process. There are different types of DNA sequencers out there, but most of them work on the principles of nucleotide compatibilities and nucleotide labeling and detecting as described above. These high-throughput sequencing technologies parallelize the sequencing process, allowing the production of thousands or millions of sequences concurrently. Some examples of these methods are 454, SOLiD and Illumina platforms.
Here at Basepaws, we use the Illumina DNA sequencing platforms - one of the most broadly DNA sequencers used in the labs today. This sequencing method is based on reversible dye-terminators that enable the identification of single bases as they are introduced into DNA strands. The biggest disadvantage to this method is that it can only sequence fragments that are max. 50-200 bp long. Sequencing larger segments than this greatly decreases the accuracy of the method.
3) Third-generation DNA sequencers are the most recent DNA sequencers. These techniques measure the addition of nucleotides to a single DNA molecule. Examples of such advanced technologies are SMRT and Oxford Nanopore. These technologies are predicted to take over the widely used NGS platforms in the future.
4. Sequence analysis and interpretation
With the help of Illumina sequencing platforms, we sequence 150 to 200 bp long DNA fragments from the feline samples. We can’t sequence longer chunks than this because we want to keep the sequencing method as accurate as possible. In the next step, we align these sequences according to the reference genome. Both purebred cats and mixed-breed cats sequences are included to increase the accuracy of our methodology. We have to look into each 150 bp fragment and match it to the corresponding region in the reference genome. When we match these fragments to the reference, we can notice if the sequence is the same or if there are differences in the nucleotide order. If we find differences, we can mark that on a specific position; a specific cat has a certain variant (i.e. a G instead of a C). This is how we build your cat’s genotype. We use the reference genome as a skeleton to arrange your cat’s fragments accordingly and detect the variants in the specific positions of interest. These variants, these differences are what tells us about the traits of the animal. Based on this comparison we report the sample to breed indexes, wildcat indexes, diseases and other traits.
5. How and why we are building our own database?
We are building our database based on the publicly accessible data as well as our own proprietary data. We sequence some of the sample that we collect via whole genome sequencing and store it in our database. Our aim is to build a database that will contain both genotype and phenotype information about as many cats as possible. While we’re collecting the genetic data, we are also hoping to include the phenotype traits collected from the cat owners. The more data we have on our disposal, the more we can learn about the feline ancestry, evolution, lineage and health. We want to contribute to the discovery of unknown traits of our favorite pets, and in order to do this, we need a lot more information than the research world currently has on domestic cats. We need to discover as many variants as possible and make sense of these findings. Ideally, we would want every and each kitty’s voice to be heard. Every single piece of information we gather today will benefit all the cats’ futures tomorrow.
Still curious to know more about our life in the lab? Watch this video:
Frequently asked questions
1. How have you built your database of cats of the various breeds?
We build our database using the data both in the public database as well as our proprietary data. Some of the samples we collected have been sequenced using whole genome sequencing and added to the database.
2. How do you verify that a sample cat in your database is a member of a specific breed?
First, we ask a breeder to provide us a copy of the breed certificate. We reach out to the breeder and check again about the specific cat. We also sometimes require official proof from TICA or CFA. We plan to work more closely with breeder groups in the future when we have big enough data sets and feel we can offer the breeders an additional tool as part of their assessment.
3. How many cats of each breed are in your data set?
It really varies between breeds and depends on our work with those specific breeders. We are always adding more cats and more breeds, and are planning to have the largest such database of cat breeds available to use. Right now we are focusing on adding Russian Blue, Norwegian Forest, and Munchkin samples, as well as about a dozen more.
4. Are you doing anything to ensure that the sampled cats represent a good cross-section of the different bloodlines in that breed, either in the US or worldwide?
We are hoping to add multiple bloodlines and are working with breeders in the US and worldwide to help us create a database what representative is, but we can do better. Right now we heavily lean to US breeders but plan to add more from other parts of the world. This is very breeds-specific; some breeds are well represented within the US population and some need additional samples from other parts of the world.
5. Are you working with any breed groups or cat registries?
We work with TICA and individual breeders. Our work with breeders is just getting started, and we offer free tests to breeders who have lines we still need. Please get in touch with us at firstname.lastname@example.org if you are a breeder and want to help us.
6. If you can develop useful tests for genetic traits, will this information be freely shared with the research community or do they intend to develop proprietary tests?
We plan to share our findings with the whole cat community!
We hope that this insight behind the scenes at Basepaws gave you a clearer picture of our mission. Do you have more questions? Drop us an email to email@example.com