uSort-M

Rapid and low-cost parsed variant library generation

uSort-M converts pooled DNA libraries into large collections of individually-isolated, sequence-verified variants at a fraction of traditional gene synthesis costs.

Overview

Traditional approaches to generating parsed variant libraries require expensive per-gene synthesis and individual cloning. uSort-M uses FACS to isolate single cells from a pooled transformation, then identifies variants by amplicon sequencing with well-specific barcodes.

Key advantages:

• Significant cost savings compared to traditional gene synthesis
• 10-day working time from oligo pool to verified clones
• Scalable from tens to thousands of variants
• Compatible with diverse library inputs

Installation

# Basic installation
pip install -e .

# Full installation with all dependencies
pip install -e ".[all]"

External Tools (for demultiplexing)

The usortm demux command requires these tools installed separately:

Tool	Min Version	Purpose	macOS	Linux
dorado	1.3+	Barcode demultiplexing	GitHub releases (.zip)	GitHub releases (.tar.gz)
minimap2	2.20+	Reference alignment	brew install minimap2	apt/dnf install minimap2
samtools	1.16+	BAM processing & consensus	brew install samtools	apt/dnf install samtools

Windows users should run inside WSL2 (Ubuntu) and use the Linux instructions. usortm auto-discovers dorado in ~/Downloads/dorado-*/bin/ and ~/.dorado/bin/; set DORADO_PATH, MINIMAP2_PATH, or SAMTOOLS_PATH to override.

Quick Start

Estimate costs

usortm estimate --library-size 500 --seq-length 300

Plan and execute a full workflow

# 1. Initialize project from variant list
usortm plan variants.csv --output my_project/

# 2. [Perform wet lab: assembly, sorting, barcoding, sequencing]

# 3. Process sequencing data (with library CSV for variant calling)
usortm demux my_project/ --fastq sequencing-data.fastq --library-csv variants.csv

# 4. Generate hit-picking list
usortm pick my_project/

# 5. Create final report
#    Generates HTML summary, CSVs, and a shareable zip file
usortm report my_project/

CLI Commands

Command	Description
estimate	Quick cost and effort estimation
plan	Initialize project from variant list
demux	Demultiplex sequencing data (LevSeq barcodes via dorado, reference alignment, consensus, variant calling)
pick	Generate Integra ASSIST hit-picking list (ordered by input library)
reorder	Export synthesis order for dropout variants (unrecovered after round 1)
merge	Merge hit-picking lists from multiple rounds into a single final pick list
report	Generate final plate maps, coverage stats, HTML summary, and shareable zip

Multi-round workflow (recovering dropouts)

After round 1 pick, variants that were not recovered (dropouts) can be re-synthesized and run through a second round of uSort-M to maximize library coverage.

# After round 1 pick completes, export a synthesis order for dropout variants
usortm reorder my_project/

# [Re-synthesize dropouts and perform wet lab for round 2]

# Plan round 2 against the existing project
usortm plan dropouts.csv --output my_project/ --round 2

# Demultiplex round 2 sequencing data
usortm demux my_project/ --fastq round2-data.fastq --round 2

# Pick round 2 hits
usortm pick my_project/ --round 2

# Merge round 1 and round 2 picks into a single final list
usortm merge my_project/

# Generate the merged report (covers both rounds)
usortm report my_project/ --round merged

After usortm merge, the combined Integra ASSIST pick list is written to my_project/merged/pick/Integra ASSIST Input/. Variants are placed at their library-ordered positions across both rounds, with round 2 hits filling in wherever round 1 did not recover.

Example: Cost Estimate

usortm estimate -n 500 -l 300

# Output:
# ╭────────────────────────────────╮
# │ uSort-M Cost Estimate          │
# │ Library: 500 variants × 300 bp │
# ╰────────────────────────────────╯
#
#                   Cost Breakdown
# ╭────────────────────────┬─────────┬─────────────╮
# │ Step                   │ uSort-M │ Traditional │
# ├────────────────────────┼─────────┼─────────────┤
# │ Synthesis              │  $1,373 │     $17,500 │
# │ Cloning                │     $54 │      $6,048 │
# │ Sorting                │    $104 │         N/A │
# │ Barcoding + Sequencing │  $1,477 │        $500 │
# │ Hit-picking            │     $80 │         N/A │
# │ Total                  │  $3,088 │     $24,048 │
# ╰────────────────────────┴─────────┴─────────────╯
#
#   7.8-fold savings with uSort-M

Workflow Timeline

Day	Step	Duration
1	Pooled assembly + transformation	4-6 hours
2+	FACS sorting	~8 min/plate
2+	Outgrowth	Overnight
3+	PCR barcoding	~50 min/plate
4-6	Sequencing	1-3 days
6+	Analysis + hit-picking	1-2 hours

Python API

from usortm.costs import cost_functions as cf

# Calculate costs
costs = cf.usortm_total_cost(
    library_sizes=[500, 1000],
    seq_lengths=[300]
)

# Run coverage simulations
from usortm.simulate import sortm

results = sortm.sortm(
    n_sims=1000,
    lib_size=500,
    skew=4,
    fold_sampling=8,
)

Documentation

Full documentation available at fordycelab.github.io/usortm

Citation

If you use uSort-M in your research, please cite:

Olivas MB, Almhjell PJ, Shanahan JD, Fordyce PM. uSort-M: Scalable isolation 
of user-defined sequences from diverse pooled libraries. bioRxiv (2026). DOI: 10.64898/2026.01.12.699065

License

MIT License - see LICENSE for details.

Links

• GitHub Repository
• Documentation
• Fordyce Lab