Generate Array of Alerts Warnings to be Associated with Each Primer

[David-Bianchi]

As a user, I would like to generate an array-like structure of alerts that is associated with each primer to be output to the primer output file for all generated primers.

The list of alerts are as follows:

1. High GC or High AT regions

• Example: "GCGCCCCC" or "AATTTAAAA"

2. Homopolymer stretches (> 5bp), like a polyA tail etc.

• Example: "AAAAA"

3. No repeated fragments (> 16 bp)
4. High GC Content (> 70%)
5. Very low or Very High Tm values (60-75 deg. C) (confirm with Nilmani the range for this), and see features prioritization sheet: https://docs.google.com/spreadsheets/d/16XHy-faOaQ7RQPW3zCwIzpDXPglADraUQ3HerBFSgx8/edit?gid=327078375#gid=327078375
   Results based alert:
6. Temperature difference (**> 5 deg. C): The difference between forward and reverse primers should be within this temperature difference.

These alerts should be written as an additional column to the output.csv generated by the CLI such that it can then be read and displayed by the front-end.

Example Design visualization of how alerts will appear in the front-end when they are passed from the back-end:

See column "Alerts"

Modules to use:
Biopython methods?

List of ACs:
AC1. For CLI display warnings in the terminal AND write to text file
AC2. For front-end write the warnings to .json

Sample Inputs/Outputs:
Sample Results.csv, where we would check for any alerts: https://github.com/Zhao-Group/Primer_Design_and_Worklists/blob/hardcode_to_args/Primer_Design/Primers_HMT_Plate2/HMT_Designed_primers.csv

Example alerts that would be generated (e.g.):

• Phy_N227S_Fwd - Tm below 60/Tm out of range (the Tm is 46C)

[David-Bianchi]

@David-Bianchi @avireusdev11

Other possible alert "Hairpin Formation", check if we have a method for doing this or if we need to make another issue for implementing one of these methods.

[avireusdev11]

Check which method to use to calculate Tm values. Biopython provides 3 methods which can be seen in the docs at http://biopython.org/docs/1.75/api/Bio.SeqUtils.MeltingTemp.html

[David-Bianchi]

@avireusdev11 we'll have to check what version of primer3 we want to be using, I thought we are using this one from requirements.txt

But I'm confused because it says there's a deprecation of .calcHairpin() since v1.0?: https://libnano.github.io/primer3-py/api/bindings.html

I thought maybe it should be calc_hairpin() instead like shown here: https://pypi.org/project/primer3-py/

Maybe just a weird labeling thing haha, because looks like it is working fine, but we can double check. Thanks for including the link out in the function header, that's great!

[David-Bianchi]

@avireusdev11 we'll also add "20%" low GC alert, based on Nilmani's suggestion, by default.