1. Before You Screen

Resources

We highly recommend reviewing the following materials before screening:

Feldman, D., Funk, L., Le, A. et al. Pooled genetic perturbation screens with image‑based phenotypes. Nat Protoc 17, 476–512 (2022). doi.org/10.1038/s41596-021-00653-8
Walton, R. T., Singh, A., Blainey, P. C. et al. Pooled genetic screens with image‑based profiling. Mol Syst Biol 18, e10768 (2022). doi.org/10.15252/msb.202110768
Feldman, D., Singh, A., Schmid‑Burgk, J. L. et al. Optical pooled screens in human cells. Cell 179, 787–799.e17 (2019). doi.org/10.1016/j.cell.2019.09.016
Blainey Lab OPS Protocols. Note: Ignore the GitHub Repository as this is outdated!

OPS Data Collection

Data Structure

During a brieflow run, microscope files are loaded from datafames (SBS/phenotype samples dfs) that specify a file’s path and metadata (plate, tile, well, etc). The file paths and metadata are parsed from a data directory using a regex expression. See the 0.configure_preprocess_params.ipynb notebook for more information on how this works.

With this setup, data can be saved anywhere with any format. But we recommend:

saving in a safe (protected) location outside of the brieflow-analysis directory such that one can distinguish between inputs and intermediate outputs
the following hierarchical structure and naming conventions for simplicity:

Hierarchical Structure

screen_name/
└── plate_1/
    ├── input_sbs/
    │   ├── c1/
    │   ├── c2/
    │   └── ... (cycles of sequencing data)
    └── input_ph/
        ├── round_1/
        ├── round_2/
        └── ... (rounds of phenotyping data)
├── plate_2/
└── ... (plates of pooled screening data)

SBS (Sequencing) Files

Files should be located within cycle directories (c1, c2, etc.)
Naming format should include plate number, well ID, and tile number information
Channel information should be included if each ND2 image is for an individual channel. Otherwise, channel information can be included, but is not necessary.
When generating SBS data, one can include additional cycles or channels that assist with segmenting cells. Brieflow’s preprocessing and SBS modules have methods for using these additional cycles/channels.

Example SBS filename: screen_name/plate_1/c2/P001_Wells-A3_Points-214.nd2

Phenotype Files

Files should be located within round directories (round_1, round_2, etc.)
Naming format should include plate number, well ID, and tile number information
Channel information should be included if each ND2 image is for an individual channel. Otherwise, channel information can be included, but is not necessary.

Example Phenotype filename: screen_name/plate_1/input_ph/round_1/P001_Wells-B1_Points-585.nd2

Common Pitfalls

Here are some common pitfalls wtih OPS data collection and how to avoid them.

Alignment Verification

Critical Quality Control Step: Check alignment between:

Rounds of phenotyping data (round_1 vs. round_2 vs. …)
Cycles of sequencing data for each cycle (c1 vs. c2 vs. …)

Alignment verification should be performed before proceeding with downstream analysis. Downstream analysis will be impossible with large spatial shifts in alignment.