BioFoundry Project at the HMC BioMakerspace. An automated workflow to design primers for circularised DNA and for FastCloning experiments. This workflow supports fasta and genbank DNA file format as well as string format for plasmid sequence inputting to allow better user experience. For designing FasctClong Primers, we thank NEB Tm Calculator for making their primer melting/annealing temperature tools publicly available.
Install dependencies with
pip install -r requirements.txt
In addition, you need to have a Google Chrome browser installed on your device (preferrably a Macbook). Check the version of your Chrome browser by clicking on Help>About Google Chrome, then download the Chrome webdriver here that corresponds to your own Chrome version. Move the Chrome webdriver you installed to the Biofoundry repository, and you should be ready to go.
If you would like to install them manually:
- Primer3-py (Download via terminal with
pip install primer3-py) - Pandas (Download via terminal with
pip install pandasorconda install pandas) - selenium (Download via terminal with
pip install selenium)
Here is a super short tutorial video for this repo, especially on how to corroborate the primers designed by the workflow. Note that Benchling crashed in the middle of the video ahaha :-)......
Clone or download this repository, enter the local directory where the repo is located. If you haven't already, install the dependencies with
pip install -r requirements.txt
More accessible installation methods to come later.
Enter ipython in your terminal before you design your primer. Then do the following:
run fastCloningPrimer.py
If you do not prefer to use ipython, you can instead directly do the following in the terminal:
(1) enter the Biofoundry directory, type python to enter python mode
(2) type from fastCloningPrimer import * to import all functions.
This repository enables two types of primer designs. You can either design primer pairs for simply isolating an area from a plasmid; or you can design primer pairs for FastCloning experiments.
- You can simply design primer3 primer pairs for simply isolating an area from a plasmid. The output will be a csv file. You can choose to have your primers to be outputted in a benchling acceptable format or not. Check out the sample outputs for this part in files
sampleOutputs/plasmidPrimerInfo.csvandsampleOutputs/benchlingPlasmidPrimerInfo.csv.
You have two options. You can either input the plasmid sequence as a string or as a fasta/genbank file. Note that only fasta/genbank formats are supported for now.
Note that the goal sequence (goalSeq), or the sequence you would like to isolate from the plasmid, always needs to be inputted as a string.
(1) If you input the plasmid sequence as a string,
plasmidPrimers(plasmidSeq, goalSeq, benchling=True, destinationAddress='plasmidPrimerInfo.csv', benchlingAddress='benchlingPlasmidPrimerInfo.csv', primerOptTm=PRIMER_OPT_TM, primerMinSize=PRIMER_MIN_SIZE, enzyme="Taq", maxTempDiff=MAX_TEMP_DIFF)
# Note that destinationAddress, benchlingAddress, and benchling are unnecessary. You can choose to change the input. If you choose not to, you can simply do the following:
fastCloningPrimersFile(vectorPlasmidAddress, insertPlasmidAddress, vectorSeq, insertSeq). The outputs can be found at the default addresses (destinationAddress='plasmidPrimerInfo.csv', benchlingAddress='benchlingPlasmidPrimerInfo.csv').
# Outputs friendly for Benchling will be automatically generated. If you prefer to not output benchling format files, please set the benchling boolean to False.
# primerOptTm (optimal primer temperature) and primerMinSize (shortest acceptable primer length) are the two primer3 parameters that you can adjust, along with the maxTempDiff, which is the maximum melting temperature difference between two primers in each primer pair.
# You can choose which enzyme you are using. Currently we support Taq polymerases OR NEB phusion polymerase. You can input that as either `enzyme="Taq"` or `enzyme="phusion"`.
## TEST INPUTS:
## plasmidPrimers(vectorPlasmidSeq1, vectorSeq1)
## plasmidPrimers(insertPlasmidSeq1, insertSeq1)
This gives you several primer pairs for isolating insertSeq1 from its plasmid insertPlasmidSeq1.
(2) If you input the plasmid sequence as a fasta/genbank file,
plasmidPrimers(plasmidSeqFile, goalSeq, benchling=True, destinationAddress='plasmidPrimerInfo.csv', benchlingAddress='benchlingPlasmidPrimerInfo.csv', primerOptTm=PRIMER_OPT_TM, primerMinSize=PRIMER_MIN_SIZE, enzyme="Taq", maxTempDiff=MAX_TEMP_DIFF)
# Note that destinationAddress, benchlingAddress, and benchling are unnecessary. You can choose to change the input. If you choose not to, you can simply do the following:
fastCloningPrimersFile(vectorPlasmidAddress, insertPlasmidAddress, vectorSeq, insertSeq). The outputs can be found at the default addresses (destinationAddress='plasmidPrimerInfo.csv', benchlingAddress='benchlingPlasmidPrimerInfo.csv').
# Outputs friendly for Benchling will be automatically generated. If you prefer to not output benchling format files, please set the benchling boolean to False.
# primerOptTm (optimal primer temperature) and primerMinSize (shortest acceptable primer length) are the two primer3 parameters that you can adjust, along with the maxTempDiff, which is the maximum melting temperature difference between two primers in each primer pair.
# You can choose which enzyme you are using. Currently we support Taq polymerases OR NEB phusion polymerase. You can input that as either `enzyme="Taq"` or `enzyme="phusion"`.
## TEST INPUTS:
## plasmidPrimersFile(vectorPlasmid1AddressFA, vectorSeq1)
## plasmidPrimersFile(insertPlasmid1AddressGB, insertSeq1)
- You can design primer3 primer pairs for your fastCloning experiments. Check out the sample outputs for this part in files
sampleOutputs/fastCloningPrimerInfo.csvandsampleOutputs/benchlingfastCloningPrimerInfo.csv.
Again, you have two options. You can either input the plasmid sequence as a string or as a fasta/genbank file. Note that only fasta/genbank formats are supported for now.
Note that the goal sequence (goalSeq), or the sequence you would like to isolate from the plasmid, always needs to be inputted as a string.
(1) If you input the plasmid sequences as strings,
fastCloningPrimers(vectorPlasmidSeq, insertPlasmidSeq, vectorSeq, insertSeq, maxTempDiff=MAX_TEMP_DIFF, destinationAddress='fastCloningPrimerInfo.csv', benchlingAddress='benchlingfastCloningPrimerInfo.csv', benchling=True, primerOptTm=PRIMER_OPT_TM, primerMinSize=PRIMER_MIN_SIZE)
# Note that destinationAddress, benchlingAddress, and benchling are unnecessary. You can choose to change the input. If you choose not to, you can simply do the following:
fastCloningPrimersFile(vectorPlasmidAddress, insertPlasmidAddress, vectorSeq, insertSeq). The outputs can be found at the default addresses (destinationAddress='plasmidPrimerInfo.csv', benchlingAddress='benchlingPlasmidPrimerInfo.csv').
# Outputs friendly for Benchling will be automatically generated. If you prefer to not output benchling format files, please set the benchling boolean to False.
# primerOptTm (optimal primer temperature) and primerMinSize (shortest acceptable primer length) are the two primer3 parameters that you can adjust, along with the maxTempDiff, which is the maximum melting temperature difference between two primers in each primer pair.
# You can choose which enzyme you are using. Currently we support Taq polymerases OR NEB phusion polymerase. You can input that as either `enzyme="Taq"` or `enzyme="phusion"`.
## TEST INPUTS:
## fastCloningPrimers(vectorPlasmidSeq1, insertPlasmidSeq1, vectorSeq1, insertSeq1)
Check out your destinationAddress for your primers.
(2) If you input the plasmid sequences as fasta/genbank files (please make sure two plasmid files have the same format),
fastCloningPrimersFile(vectorPlasmidAddress, insertPlasmidAddress, vectorSeq, insertSeq, maxTempDiff=MAX_TEMP_DIFF, destinationAddress='fastCloningPrimerInfo.csv', benchlingAddress='benchlingfastCloningPrimerInfo.csv', benchling=True, primerOptTm=PRIMER_OPT_TM, primerMinSize=PRIMER_MIN_SIZE)
# Note that destinationAddress, benchlingAddress, and benchling are unnecessary. You can choose to change the input. If you choose not to, you can simply do the following:
fastCloningPrimersFile(vectorPlasmidAddress, insertPlasmidAddress, vectorSeq, insertSeq). The outputs can be found at the default addresses (destinationAddress='plasmidPrimerInfo.csv', benchlingAddress='benchlingPlasmidPrimerInfo.csv').
# Outputs friendly for Benchling will be automatically generated. If you prefer to not output benchling format files, please set the benchling boolean to False.
# primerOptTm (optimal primer temperature) and primerMinSize (shortest acceptable primer length) are the two primer3 parameters that you can adjust, along with the maxTempDiff, which is the maximum melting temperature difference between two primers in each primer pair.
# You can choose which enzyme you are using. Currently we support Taq polymerases OR NEB phusion polymerase. You can input that as either `enzyme="Taq"` or `enzyme="phusion"`.
## TEST INPUTS:
## fastCloningPrimersFile(vectorPlasmid1AddressFA, insertPlasmid1AddressFA, vectorSeq1, insertSeq1)
## fastCloningPrimersFile(vectorPlasmid1AddressGB, insertPlasmid1AddressGB, vectorSeq1, insertSeq1)
Check out your destinationAddress for your primers.
- Allow the primer design with phusion for plasmid primer designs and fastCloning primers design, using Serenium webscraping of the NEB Tm Calculator.
- Allow the users to manually decide primer3 parameters 'primerOptTm (optimal primer temperature) and primerMinSize (shortest acceptable primer length)'.
- Allow outputs that are in Benchling readable format. If users would like to import the primer outputs from this workflow to benchling, simply click on
import Oligoon benchling. Copy paste the primer information in your benchling-compatible output files and paste them into the textbox in benchling.
Currently, this workflow supports primer-3 supported primer design for Taq polymerase. For Phusion polymerases, this workflow designs primers by finding Taq primers in a range of temperatures around the optimal melting temperature of phusion, and then cross-checking whether these primers work through webscraping at the NEB Tm Calculator.
This method, while mostly reliable, depends on the fact that Taq and Phusion have similar temperature. While this is mostly true, there are cases where this is not true. A more straightforward approach would be find the melting temperature calculation formula for Phusion and put that into primer3 to find primers directly for Phusion, instead of what we are currently doing.
In order to achieve this, functions you should focus on in our existing codebase include:
vectorPrimerDesign
insertPrimerDesign
And the wrapper functions.
You should find formulae and parameter values in resources and papers provided by roya; look for places to insert these formulae and values in the primer3 manual, and then revise our current methods to enable our desired functionalities.
Developers:
Tom Fu (@tommyfuu)
Richard Chang (@richanghmc)
Affiliation: Harvey Mudd College BioMakerspace (Polymerspace).
Faculty advisor: Dr. Dan Stoebel.
This project is part of the intellectual property of the Harvey Mudd College BioMakerspace. Please notify the space at tfu@g.hmc.edu for consent before using any of the materials in this repository. Please cite us if you use our repository for academic sharing purposes.
The funding support of the Harvey Mudd College BioMakerspace comes from Harvey Mudd College Office of Community Engagement and the college's Shanahan Fund.
We also thank Zhengyao Lin for his assistance on the web scraping part of the project.