split-oligo-designer

Overview

This repository contains the code to design the oligonucleotides for mFISH3D.

The code is heavily relying on OligoMiner tool.

Software prerequisites

Tested on Ubuntu 20.04 LTS with the following version of software.

• BLAST (2.13.0)
• Python 3.8

Make sure to set the path to the BLAST.

Installing dependencies

1. Make sure you have conda installed.
2. Create a new environement (python=3.8), and install the dependencies.

$ git clone https://github.com/tatz-murakami/split-oligo-designer.git
$ cd split-oligo-designer
$ conda env create -f environment.yml
$ conda activate oligo

$ pip install . # install oligodesigner

Database and template sequence

Make database

1. Download and make the database for blast. The database for the cDNA can be found at Ensembl
2. Generate the blast database according to the instruction of NCBI. example: ./data/human_transcriptome

Download fasta template

Download the fasta template from NCBI or else. example: ./data/slc17a7/slc17a7_hs.fasta

Example usage

The jupyter notebook is included OligoDesign.ipynb.

Parameters

The example of the parameters are shown below.

mFISH3D_param = {
    'fasta':'/home/tmurakami/src/split-oligo-designer/data/slc17a7/slc17a7_hs.fasta',
    'database':'/home/tmurakami/src/split-oligo-designer/data/human_transcriptome/human_transcriptome_db',
    'minimum_offtarget_gap':100,
    'hcr_seqs':{
            'seq_even_l':'GAGGAGGGCAGCAAACGGaa',
            'seq_odd_r':'atGAAGAGTCTTCCTTTACG',
            'seq_even_r':'',
            'seq_odd_l':''
        },
    'self_remove': True # set this True if your template sequence appears in database.
}

minimum_offtarget_gap If the gap between two non-specific binding is more than minimum_offtarget_gap, the pair is not regarded to cause a off-target signal. Recommended value: 100.

hcr_seqs The sequences of HCR fragments. The example design can be found in ./oligodesigner/parameters.py

self_remove Set self_remove True if you want to remove the template sequence from off-target analysis. Otherwise, the gene of your interest could be regarded as an off-target product. Turn this to False when your template is not found in database (e.g. GFP).

The code requires the parameters for OligoMiner. The example is below.

oligominer_param = {
    'l':20,
    'L':20,
    'gcPercent':25,
    'GCPercent':75,
    'tm':20,
    'TM':100,
    'X':'AAAAAA,TTTTTT,CCCCCC,GGGGGG',
    'sal':390,
    'form':30,
    'sp':1,
    'concA':25,
    'concB':25,
    'headerVal':None,
    'bedVal':False,
    'OverlapModeVal':False,
    'verbocity':False,
    'reportVal':True,
    'debugVal':False,
    'metaVal':False,
    'outNameVal':None,
    'nn_table':'DNA_NN3'
}

Citation

Murakami and Heintz. Multiplexed and scalable cellular phenotyping toward the standardized three-dimensional human neuroanatomy. bioRxiv (2022) https://doi.org/10.1101/2022.11.23.517711

Beliveau, B.J., Kishi, J.Y., Sasaki, H.M. et al. OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes. PNAS (2018) https://doi.org/10.1073/pnas.1714530115

License

We provide this open source software without any warranty under the MIT license.