CoMPoSE custom probe design

Purpose

Workflow for custom probe design for transgenes to be spiked into single cell probe-based detection with 10x flex. Requires input FASTA of target sequences. These are used to nominate 50 bp probe binding sites meeting sequence composition constraints. Scressn the resulting 25 bp lfet and right had arms agains the human (or mouse if modified) transcriptome for off-target hybridization. Selects non-overlapping sets to construct an IDT pool. Includes optional functions to restrict sequences or plot qc figures.

Author

Written by Jason Swinderman Reviewed by Aidan Winters Revised and error-tested with Claude Opus 5

Date

Written November, 8th 2023
Last revised: October, 6th 2026

Contents

probe_design.py              nomination, off-target handling, selection, oPool construction
probe_design_utils.py        input helpers 
probe_design_qc.py           QC plots
probe_design.ipynb           worked examples for domain and gapfill ORF probe sets in the paper
scripts/
  make_hs_refseq_blastdb.sh  build the human RefSeq RNA BLAST database
  probe_blast.sh             BLAST probe arms and filter by alignment length
references/                  input tables and third-party reference files
probe_nomination/            pipeline outputs
opools/                      IDT order sheets

Requirements

conda env create -f environment.yml
conda activate compose-probe-design

Reference files you need to download

1. Chromium Human Transcriptome Probe Set v1.0.1 (GRCh38-2020-A), from the 10x Genomics support site. Place it at references/Chromium_Human_Transcriptome_Probe_Set_v1.0.1_GRCh38-2020-A.csv — this is the default path in gene_to_ensembl_wta_filter() and construct_probe_reference(). https://www.10xgenomics.com/support/software/cell-ranger/downloads

2. Human RefSeq RNA BLAST database:

   ./scripts/make_hs_refseq_blastdb.sh -o references

NCBI BLAST of RHS and LHS probe candidates against reference transcriptome

lhs_rhs_probe_binding_site_split()

• Constructs FASTA of the nominated probe-binding sites for the lhs and rhs sequences. 
• This is the input for the below shell script(s). It's easiest to run this in commandline. 

make_hs_refseq_blastdb.sh

• Constructs a human refseq blast database 

domain_probe_blast.sh

• BLASTs nominated LHS and RHS probe binding sites and records those with more than 20 bp recognition of sequences in the reference transcriptome

Filter off-target binding probes from BLAST results

read_blast_results()

• imports and subsets blast off-target hits annotating lhs or rhs probes with >20 offtarget priming 
• annotates each probe with the maximum off-target gene 

process_lhs_rhs()

• concatenates rhs and lhs off-target binding infromation from blast with the probe annotation table from nominate_probes()

Nominating top sets of probes for each target.

nominate_top_probe_set() calls:

• calculate_priority_score() to calculate priority score for each probe pair 
• nominate_non_overlapping_sets() to generate all unique sets of nonverlapping probe pairs
• select_highest_priority_sets() to assign the set of probe-pairs with the greatest total priority score. 

Citation

Scalable probe-based single-cell transcriptional profiling for virtual cell perturbation mapping and synthetic biology phenotyping Jason T. Swinderman, Po-Yuan Tung, Aidan Winters, Laine Goudy, Caroline M. Wilson, Lexi R. Bounds, Noam Teyssier, Ayush Agrawal, Alex Dobin, Tony Hua, Hani Goodarzi, Felix Y. Feng, Alex Marson, Dave P. Burke, Patrick D. Hsu, Yusuf H. Roohani, Silvana Konermann, Michael Kosicki, Nianzhen Li, Luke A. Gilbert bioRxiv 2026.02.04.703058; doi: https://doi.org/10.64898/2026.02.04.703058

License

MIT License

Copyright (c) 2026 Jason Swinderman

Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions:

The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software.

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