Quick Start Guide

This guide will get you up and running with U-Probe in just a few minutes. We'll walk through a simple probe design workflow from start to finish.

Prerequisites

Before you begin, make sure you have:

• U-Probe installed (see installation)
• A genome FASTA file
• A gene annotation GTF file
• Basic knowledge of YAML configuration files

Your First Probe Design

Let's design probes for some target genes using a simple configuration.

Step 1: Prepare Your Data

You'll need two types of files:

1. Genome files (FASTA + GTF)
2. Configuration files (YAML)

For this tutorial, we'll assume you have:

• /path/to/genome.fa - Genome FASTA file
• /path/to/annotation.gtf - Gene annotation GTF file

Step 2: Create Configuration Files

Create genomes.yaml:

# genomes.yaml
human_demo:
  description: "Demo human genome"
  species: "Homo sapiens"
  fasta: "/path/to/genome.fa"
  gtf: "/path/to/annotation.gtf"
  align_index:
    - bowtie2
    - blast
  jellyfish: false

Create protocol.yaml:

# protocol.yaml
name: "MyFirstProbes"
genome: "human_demo"

# Target genes to design probes for
targets:
  - "GAPDH"
  - "ACTB"
  - "TP53"

# How to extract target regions
extracts:
  target_region:
    source: "exon"        # Extract from exons
    overlap: 10           # Overlap between adjacent extracts
    length: 120           # Length of each target region

# Probe design specifications
probes:
  main_probe:
    template: "{spacer}{target_binding}{barcode}"
    parts:
      spacer:
        expr: "'TTTTTTTTTT'"
      target_binding:
        length: 25
        expr: "rc(target_region[0:25])"
      barcode:
        length: 15
        expr: "encoding[target]['BC1']"

# Barcode sequences for each gene
encoding:
  GAPDH:
    BC1: "ACGTACGTACGTACG"
  ACTB:
    BC1: "TGCATGCATGCATGC"
  TP53:
    BC1: "CGATCGATCGATCGA"

# Quality control attributes
attributes:
  gc_content:
    target: main_probe
    type: gc_content
  melting_temp:
    target: main_probe
    type: annealing_temperature

# Filtering criteria
post_process:
  filters:
    gc_content:
      condition: "gc_content >= 0.4 & gc_content <= 0.6"
    melting_temp:
      condition: "melting_temp >= 50 & melting_temp <= 65"

Step 3: Run the Complete Workflow

Now run U-Probe with a single command:

uprobe run \
  --protocol protocol.yaml \
  --genomes genomes.yaml \
  --output results/ \
  --threads 4 \
  --raw

This command will:

1. Build genome indices (if needed)
2. Validate your target genes
3. Extract target regions
4. Design probes
5. Calculate quality attributes
6. Apply filters
7. Save results to CSV files

Step 4: Examine the Results

Check the results directory:

ls results/
# Output:
# MyFirstProbes_20240131_143022.csv      # Filtered probes
# MyFirstProbes_20240131_143022_raw.csv  # All probes (if --raw used)

The CSV files contain your designed probes with all calculated attributes:

target,target_region,main_probe,gc_content,melting_temp,passed_filters
GAPDH,ATGC...,ACGT...,0.52,58.3,True
ACTB,CGTA...,TGCA...,0.48,55.7,True
...

Step-by-Step Workflow

For more control, you can run individual steps:

Step 1: Build Genome Index

uprobe build-index \
  --protocol protocol.yaml \
  --genomes genomes.yaml \
  --threads 4

Step 2: Validate Targets

uprobe validate-targets \
  --protocol protocol.yaml \
  --genomes genomes.yaml

Step 3: Generate Target Sequences

uprobe generate-targets \
  --protocol protocol.yaml \
  --genomes genomes.yaml \
  --output results/

Step 4: Design Probes

uprobe construct-probes \
  --protocol protocol.yaml \
  --genomes genomes.yaml \
  --targets results/target_sequences.csv \
  --output results/

Step 5: Post-Process (Add Attributes & Filter)

uprobe post-process \
  --protocol protocol.yaml \
  --genomes genomes.yaml \
  --probes results/constructed_probes_combined.csv \
  --output results/ \
  --raw

Understanding the Output

Probe CSV Columns

The output CSV files contain these key columns:

• target: Target gene identifier
• target_region: Extracted genomic sequence
• [probe_name]: Designed probe sequence(s)
• [attribute_name]: Calculated quality metrics
• passed_filters: Whether the probe passed all filters

Quality Metrics

Common quality attributes include:

• gc_content: GC content (0.0 to 1.0)
• annealing_temperature: Melting temperature (°C)
• self_match: Self-complementarity score
• fold_score: Secondary structure propensity
• mapped_genes: Off-target binding potential

Customizing Your Design

Probe Structure

Modify the probe template to change structure:

probes:
  forward_probe:
    template: "{primer}{target_binding}"
    parts:
      primer:
        expr: "'ACGTACGT'"  # Fixed primer sequence
      target_binding:
        length: 20
        expr: "target_region[10:30]"
  
  reverse_probe:
    template: "{target_binding}{primer}"
    parts:
      target_binding:
        length: 20
        expr: "rc(target_region[30:50])"
      primer:
        expr: "'TGCATGCA'"

Target Extraction

Change how target regions are extracted:

extracts:
  target_region:
    source: "genome"      # Extract from anywhere in genome
    length: 200           # Longer regions
    overlap: 50           # More overlap
    # Custom genomic coordinates
    coordinates:
      - "chr1:1000000-1001000"
      - "chr2:2000000-2001000"

Quality Filters

Adjust filtering criteria:

post_process:
  filters:
    # Stricter GC content
    gc_content:
      condition: "gc_content >= 0.45 & gc_content <= 0.55"
    
    # Temperature range
    melting_temp:
      condition: "melting_temp >= 55 & melting_temp <= 60"
    
    # Exclude high off-targets
    mapped_genes:
      condition: "mapped_genes <= 3"

Common Use Cases

FISH Probes

For fluorescence in situ hybridization:

probes:
  fish_probe:
    template: "{target_binding}{spacer}{fluorophore_binding}"
    parts:
      target_binding:
        length: 30
        expr: "rc(target_region[0:30])"
      spacer:
        expr: "'TTTTTT'"  # Poly-T spacer
      fluorophore_binding:
        expr: "encoding[target]['fluorophore']"

PCR Primers

For amplification-based methods:

probes:
  forward_primer:
    template: "{primer_seq}"
    parts:
      primer_seq:
        length: 22
        expr: "target_region[0:22]"
  
  reverse_primer:
    template: "{primer_seq}"
    parts:
      primer_seq:
        length: 22
        expr: "rc(target_region[-22:])"

Troubleshooting

No Targets Found

If no target sequences are generated:

1. Check gene names in your GTF file
2. Verify source parameter (exon, gene, etc.)
3. Reduce length or overlap parameters

No Probes Pass Filters

If all probes are filtered out:

1. Relax filtering conditions
2. Check attribute calculations
3. Use --raw to see all designed probes

Performance Issues

For large genomes or many targets:

1. Increase --threads parameter
2. Process targets in smaller batches
3. Use SSD storage for genome files

Next Steps

Now that you've completed your first probe design:

1. Try the AI Agent for an interactive design experience: uprobe agent
2. Explore more examples for different applications
3. Learn about advanced workflows
4. Customize your designs using the configuration guide
5. Integrate U-Probe into your pipelines with the python_api

Tip

Join our GitHub Discussions to share your designs and get help from the community!