TITLE: Designing an EpiTYPER bisulfite sequencing assay for age estimation in Acinonyx jubatus based on human orthologues
AUTHORS: Louis-Stéphane Le Clercq, Desire Dalton, Antoinette Kotze, Paul Grobler

[DESCRIPTION]
Age is key factor in animal ecology as it can be used to assign animals to important age classes, ranging from immature young to reproductive adults and eventual old age and fragility. Different groups contribute to different aspects that need to be considered when modeling current and future population dynamics as part of continued conservation efforts. Due to the need of an accurate molecular method for assigning age, several studies have explore various aspects of epigenetic clocks. Epigenetics is a collective term for mechanisms that modify DNA and DNA packaging, independent of genetic sequence. One widely studied epigenetic feature is DNA methylation; a process that adds a methyl group to the 5’ cytosine of Cytosine-Guanine pairs (CpG’s). Studies have revealed that within genes, nearly a third of all CpG sites are influenced by age. Given its consistency, the epigenetic clock is a promising avenue of chronological age prediction which has been illustrated in many human studies. This protocol illustrates how CpG's with known age-correlations from human studies can be used to (1) identify orthologous regions in other species and (2) design primers to assay differential methylation using EpiTYPER mass array technology. 


[BEFORE_START]
You need to know the CG values for the CpG's you would like to design the assay.


 


[GUIDELINES]
None

[STEPS]
SECTION: Retrieving CpG from human sequencing
1. Most research papers on humans give a "CG" value which corresponds to a CpG site in the human genome based on Illumina sequencing. The following steps were used to retrieve the human sequence for reported CG values using the University of California Santa Cruz (UCSC) Genome Browser (https://genome.ucsc.edu/).
SECTION: Finding the animal orthologues
2. The next steps are to find the orthologous gene sequence for the target species (e.g., Acinonyx jubatus) using NCBI Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
SECTION: Designing Primers for the EpiTYPER assay
3. The next steps are used to design the EpiTYPER primers on their EpiDesigner website (https://www.epidesigner.com/).
SECTION: Testing CpG coverage for selected Primers
4. The final steps are performed in R (4.0.6) using RSeqMeth (https://github.com/cran/RSeqMeth) to determine the fragmentation patterns and elucidate which CpG's can be assayed.

 

SECTION: Retrieving CpG from human sequencing
1.1.     On the landing page for the website, select "Genome Browser".
 
SECTION: Retrieving CpG from human sequencing
1.2. From the drop down menu, select the "Feb. 2009 (GRCh37/hg19)" genome build, as this is the version with the mapped CG values.
SECTION: Retrieving CpG from human sequencing
1.3. In the "position/search term" box, enter the desired CG value to look up e.g., cg00123456 and click "Go" to perform search.
 
SECTION: Retrieving CpG from human sequencing
1.4. The top of the results page will show the specific position e.g., Chr 19; 18.343,902.
SECTION: Retrieving CpG from human sequencing
1.5. The left panel of the scaffold should indicate a track for the CG value that was searched. Click on the panel (orange) for more details.

 

SECTION: Retrieving CpG from human sequencing
1.6. Click on "View DNA for this feature" and then select "Get DNA".

 

SECTION: Retrieving CpG from human sequencing
1.7. Specify to add 300-400 base pairs upstream and downstream of the CpG to ensure that the target CpG is in the middle and you have enough sequence to design primers.

 

SECTION: Retrieving CpG from human sequencing
1.8. Save the sequence in the FASTA format.
SECTION: Finding the animal orthologues
2.1. Select "Nucleotide Blast".

 
SECTION: Finding the animal orthologues
2.2. Copy and Paste the FASTA sequence into the sequence box or select the FASTA file to upload.

 
SECTION: Finding the animal orthologues
2.3. For "Database" select the "RefSeq Genome Database" and for "Organism" specify the target species e.g., Acinonyx jubatus (taxid:32536).

 
SECTION: Finding the animal orthologues
2.4. Download the FASTA for the complete aligned sequence.

 


 


 


 

SECTION: Designing Primers for the EpiTYPER assay
3.1. On the landing page, click "Start" to begin a new experiment design.
SECTION: Designing Primers for the EpiTYPER assay
3.2. Once the input page has loaded, input the target sequence in FASTA format by either copying and pasting the sequence into the box or selecting the file.

 

SECTION: Designing Primers for the EpiTYPER assay
3.3. Input the desired primer parameters or alternatively use the recommended base settings as is.
SECTION: Designing Primers for the EpiTYPER assay
3.4. Leave the Target, Excluded, and Transcription Region (Advanced) setting empty to design primers across the full sequence. Click on "Begin" to design the primers.

 
SECTION: Designing Primers for the EpiTYPER assay
3.5. The results will appear starting with an interactive diagram of the sequence, the detected CpG's, and the mapped product for each primer pair. 

 

Below the image, the primers and their details are listed for each product shown on the diagram.

 

SECTION: Designing Primers for the EpiTYPER assay
3.6. Potential primers that appear to be optimal can be selected by the left-hand tick boxes and exported in several format.
  

SECTION: Testing CpG coverage for selected Primers
4.1. Once you have downloaded RSeqMeth, open R and execute the analyses with the following code:
 
 

This is used to load the function is used to analyze the desired amplicon.

Then execute: 

 

SECTION: Testing CpG coverage for selected Primers
4.2. Six files are written to the same directory as the text file once completed, three for the analysis of "T spectra" and three for the analysis of "C spectra".

 


 

SECTION: Testing CpG coverage for selected Primers
4.3. The following results can then be viewed and assessed.