"Restrict from EMBOSS Suit version 6.3.1 with the following parameters: snucleotide1, sitelen = 4, rformat = table, enzymes = enzymes.txt. From the 4379 enzymes present in REBASE, we selected the 650 restriction enzymes that were commercially available, since this assay is meant to be used in any laboratory. From the 650 enzymes, 152 digest all P. salmonis sequences and only 65 recognized conserved restriction sites in the complete set of sequences, generating the same/similar restriction pattern (same number of bands and similar sizes)"[https://www.frontiersin.org/articles/10.3389/fmicb.2016.00643/full]
I have two sequences corresponding to the 16s rRNA gene for two strains of a certain species. I simulated the restriction digestion of these sequences using a restriction enzyme.
How do i figure out if a recognition sequence is conserved or not, if the coordinates of the cut are not the same in the sequences?
What i want to say is that they could be different for two reasons: A-not conserved or B-conserved but there was some base insertion/deletion that lead to this position mismatch.
I guess i would need some tolerance, how do i figure this tolerance value and how do i apply it?
(the sequences are flanked by the same primers motifs.)
Emboss's restrict:
One way is to align the sequences and look for the recognition sequence in both sequences. If the recognition sequence is found in both sequences, then it is conserved. However, if the recognition sequence is not found in both sequences, then it is not conserved.
Another way to figure out if a recognition sequence is conserved is to use a computer program to compare the sequences. I prefer the software BLAST.
You can also use a restriction enzyme to digest the sequences and then compare the resulting fragments. If the recognition sequence is conserved, then the fragments will be the same size in both sequences.
To determine if a recognition sequence is conserved or not, you can align the sequences and compare the regions surrounding the recognition sequence. Even if the coordinates of the cut site are not the same in the sequences, the recognition sequence itself should be conserved.
steps you can follow for this are below:
By comparing the aligned sequences and the regions surrounding the recognition site, you can determine if the recognition sequence is conserved or not.
To determine if a recognition site is conserved or not in an automated way, you would typically use a sequence alignment tool to compare the sequences of the recognition site across multiple related DNA or protein sequences.
One popular tool for performing sequence alignments is Clustal Omega, which is available online as a web tool or can be installed locally on a computer.
To use Clustal Omega, you would input the sequences of interest and select the appropriate settings for the alignment. Once the alignment is complete, you can then visually inspect the alignment to see if the recognition site is conserved across the sequences.
Additionally, you can use software tools like MEME or Multiple Em for Motif Elicitation to identify conserved motifs or recognition sites within a set of DNA or protein sequences. These tools can automatically search for conserved motifs, and provide output files that list the positions of the conserved motifs within each sequence.
Overall, the use of automated tools can greatly speed up the process of determining if a recognition site is conserved across multiple sequences.
One way to approach this problem is to use a sequence alignment tool that allows for mismatches, such as EMBOSS Needle or Clustal Omega. These tools can align the two sequences and identify conserved regions where the recognition site for the restriction enzyme is located. Once you have the alignment, you can visually inspect the alignment to identify mismatches and assess whether they are due to insertions/deletions or lack of conservation in the recognition site.
Another approach is to use a tool that can search for motifs in sequences with mismatches, such as MEME Suite or FIMO. These tools can search for a specific recognition site in both sequences, allowing for mismatches and insertions/deletions. The output will indicate whether the recognition site is present in both sequences and if it is conserved.
In terms of determining a tolerance value, this will depend on the specific restriction enzyme and the level of conservation in the recognition site. You may need to experiment with different tolerance values to determine the best approach for your specific case.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
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