Oligo Pool Coverage Calculator
Calculate oligo pool coverage and redundancy for gene synthesis, CRISPR gRNA library tiling, and capture arrays. Determine required oligonucleotides, optimal overlap spacing, and identify coverage gaps for array-based synthesis projects.
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Oligo Pool Coverage Calculator Guide
This coverage calculator determines the number of oligonucleotides required for complete target coverage in gene synthesis, CRISPR gRNA library tiling, and capture array design. Calculate optimal oligo counts, coverage redundancy, overlap spacing, and identify potential gaps for array-based synthesis projects.
Key Applications: Gene assembly (Gibson, Golden Gate), CRISPR knockout/CRISPRi libraries, exome capture arrays, oligo pool synthesis from Twist/Agilent/CustomArray, DNA data storage.
Step-by-Step Usage Guide
Step 1: Define Your Target Region
Enter the size of your target region in base pairs (bp). This could be:
- A gene or genomic region for synthesis (e.g., 5,000 bp)
- The total size of target genes for CRISPR screening (e.g., 2,000,000 bp for 1000 genes)
- A specific region for capture probe design (e.g., 500,000 bp exome)
Tip: For CRISPR libraries, calculate per gene first, then multiply by the number of genes.
Step 2: Select Coverage Strategy
Choose the strategy that matches your application:
- Tiling (Overlapping): For gene synthesis, DNA assembly, and capture arrays. Oligos overlap to ensure continuous coverage. Use Tm Calculator to verify overlap annealing temperatures.
- CRISPR gRNA Library: For genome-wide screening. gRNAs are evenly spaced across target regions. See CRISPR Library Design workflow for complete design process.
- Custom Spacing: For specialized applications requiring specific gap or overlap patterns.
Step 3: Set Oligonucleotide Parameters
Configure oligo length and spacing/overlap:
- Oligo Length: Typically 150-200 nt for gene synthesis, 20 nt for CRISPR gRNAs. Calculate properties with Molecular Weight Calculator.
- Overlap (Tiling): 20-50 bp recommended for reliable assembly. Check Secondary Structure in overlap regions.
- Spacing (CRISPR): 50-200 bp between gRNAs for optimal coverage
Step 4: Set Desired Redundancy
Redundancy determines how many times each position is covered on average:
- 1x: Single coverage - minimum cost, suitable for high-quality synthesis
- 1.5-2x: Recommended for array-based synthesis (accounts for 5-10% dropout). Estimate quality with Error Rate Calculator.
- 2-3x: For critical applications or uncertain synthesis quality. Check pool uniformity with Uniformity Estimator.
Step 5: Calculate and Review Results
Click"Calculate Coverage" to get:
- Minimum oligos required
- Actual coverage percentage
- Redundancy achieved
- Gap analysis (if any)
- Visual coverage map
Real-World Calculation Examples
Example 1: Gene Synthesis Project
Input Parameters:
- • Target Size: 10,000 bp
- • Strategy: Tiling (Overlapping)
- • Oligo Length: 200 nt
- • Overlap: 40 bp
- • Desired Redundancy: 1.5x
Results:
- • Minimum Oligos: ~94 oligos
- • Actual Coverage: 100%
- • Redundancy: 1.5x
- • Gaps: None
Interpretation: This design provides complete coverage with 1.5x redundancy, ideal for array-based synthesis. The 40 bp overlap ensures reliable assembly via Gibson or Golden Gate methods.
Example 2: CRISPR Knockout Library
Input Parameters:
- • Target Size: 2,000,000 bp (1000 genes × 2000 bp average)
- • Strategy: CRISPR gRNA Library
- • gRNA Length: 20 nt
- • Spacing: 100 bp
- • Desired Redundancy: 1x
Results:
- • Minimum gRNAs: ~16,667 gRNAs
- • Actual Coverage: 100%
- • Redundancy: 1x
- • Average: ~16.7 gRNAs per gene
Interpretation: This provides comprehensive coverage with multiple gRNAs per gene. Note: Actual usable gRNAs will be 20-40% fewer due to PAM site (NGG) availability constraints. Plan for ~10,000-13,000 usable gRNAs in final design.
Example 3: Capture Probe Array
Input Parameters:
- • Target Size: 500,000 bp (exome regions)
- • Strategy: Tiling (Overlapping)
- • Probe Length: 120 nt
- • Overlap: 60 bp (50% overlap for 2x redundancy)
- • Desired Redundancy: 2x
Results:
- • Minimum Probes: ~8,334 probes
- • Actual Coverage: 100%
- • Redundancy: 2x
- • Gaps: None
Interpretation: The 2x redundancy ensures robust capture efficiency even with synthesis dropouts. The 50% overlap provides excellent coverage for targeted sequencing applications.
Understanding Your Results
Minimum Oligos Required
This is the theoretical minimum number of oligonucleotides needed to achieve your desired coverage. For array-based synthesis, add 10-20% buffer to account for dropouts.
Formula (Tiling): ⌈(Target Size - Overlap) / (Oligo Length - Overlap)⌉ × Redundancy
Actual Coverage Percentage
The percentage of your target region that is covered by at least one oligonucleotide. 100% means complete coverage; values below 100% indicate gaps in coverage.
Redundancy
Average number of times each position is covered. Higher redundancy increases reliability but also cost. For 2025 standards:
- • 1x: Minimum for column-based synthesis
- • 1.5-2x: Standard for array-based synthesis (recommended)
- • 2-3x: For critical regions or uncertain quality
Gap Analysis
Identifies regions not covered by your design. Gaps occur when:
- • Spacing is too large relative to oligo length
- • The final oligo doesn't reach the end of the target
- • Insufficient redundancy leaves uncovered regions
Solution: Reduce spacing, increase redundancy, or manually add oligos to fill gaps.
Array-Based Synthesis Platform Guidelines
Twist Bioscience Silicon Platform (2024-2025)
- Optimal length: 150-300 nt (200 nt sweet spot, 300 nt available)
- Expected dropout: 2-5% at 200 nt (industry-leading quality)
- Synthesis error rate: 1 error per 500-1000 bp
- Recommended redundancy: 1.2-1.5x for gene synthesis (lower due to high quality)
- Overlap for assembly: 30-40 bp (Gibson), see Golden Gate details below
- Coverage example: 10 kb with 200 nt oligos, 40 bp overlap = 63 oligos base, 76-95 with 1.2-1.5x redundancy
Agilent SurePrint / CustomArray
- Optimal length: 120-200 nt (170 nt typical, quality drops >200 nt)
- Expected dropout: 15-25% at 200 nt (inkjet synthesis variability)
- Synthesis error rate: 1 error per 300-500 bp
- Recommended redundancy: 2-2.5x minimum for reliable coverage
- Overlap for assembly: 35-45 bp (compensate for higher error rate)
- Coverage strategy: Higher redundancy essential; consider reducing oligo length to 150-170 nt for better quality
CRISPR gRNA Library Design Constraints
Theoretical coverage calculations must account for PAM site availability and design rules:
PAM Site Frequency (both DNA strands):
- SpCas9 (NGG): ~12.5% genome-wide (1 in 8 bp)
- SaCas9 (NNGRRT): ~9.4% genome-wide (1 in 10.6 bp)
- Cas12a/Cpf1 (TTTV): ~6.25% genome-wide (1 in 16 bp)
Note: Single-strand NGG frequency = 6.25% (0.25 × 0.25); both strands = 12.5%
Design Constraint Filters:
- Off-target filtering: Removes 20-30% of candidates (specificity score < 0.5)
- GC content filter: Removes 10-15% (outside 40-60% range)
- Poly-T terminator: Removes 5-10% (TTTT sequences)
- Predicted efficacy: Keep top 60-70% (Doench 2016 score)
Usable gRNA Yield: From theoretical positions, expect:
- • High-stringency: 40-60% pass all filters (genome-wide screens)
- • Standard filtering: 60-80% usable (focused libraries)
- • Minimal filtering: 70-85% (preliminary screens, accept lower quality)
Reference: Doench et al. (2016) Nat Biotechnol; Hsu et al. (2013) Nat Biotechnol
Coverage Calculation Methods & Formulas
Mathematical Formulas
Coverage calculations use verified algorithms for each synthesis strategy:
Tiling Strategy Formula:
This formula accounts for overlapping regions and ensures continuous coverage.
CRISPR gRNA Library Formula:
Accounts for even spacing between gRNAs. Note: Actual designs require PAM site filtering.
Coverage Calculation:
Calculated by analyzing each position in the target region.
Synthesis Quality Parameters (2024-2025 Standards)
- Platform-Specific Dropout Rates: Twist silicon-based: 2-5% (200 nt); Agilent/CustomArray inkjet: 15-25% (200 nt); IDT column synthesis: <1% (high-fidelity). Plan redundancy based on platform choice.
- Synthesis Error Rates (peer-reviewed data): Twist silicon: 1 error per 500-1000 bp; Agilent inkjet: 1 error per 300-500 bp; IDT column: 1 error per 1000-3000 bp. Source: Kosuri & Church (2014) Nat Methods; Plesa et al. (2018).
- Assembly Method Overlap Requirements: Gibson assembly: 15-40 bp (30-40 bp optimal, Tm 48-58°C); Golden Gate: 4 bp overhang (BsaI/BbsI) or 3 bp (SapI), enzyme-specific; PCR assembly: 25-35 bp (Tm > 55°C); NEBuilder HiFi: 15-40 bp.
- CRISPR Library Tiling Density: Dense tiling (50-100 bp spacing): 10-20 gRNAs/gene for saturation mutagenesis; Standard (100-200 bp): 5-10 gRNAs/gene for knockout screens; Sparse (>200 bp): 3-5 gRNAs/gene for focused libraries.
- Capture Array Design: 1x coverage: minimum (not recommended for clinical); 2x redundancy: standard exome/panel sequencing (50% overlap tiling); 3-5x: ultra-deep coverage for variant detection, liquid biopsy applications.
Synthesis Platform Comparison (2024-2025)
Choose the optimal platform based on your coverage requirements, budget, and quality needs:
| Platform | Technology | Length Range | Dropout Rate | Error Rate | Recommended Redundancy | Best For |
|---|---|---|---|---|---|---|
| Twist Bioscience | Silicon synthesis | 150-300 nt | 2-5% | 1/500-1000 bp | 1.2-1.5× | Gene synthesis, CRISPR libraries (>5K oligos) |
| Agilent SurePrint | Inkjet synthesis | 120-200 nt | 15-25% | 1/300-500 bp | 2-2.5× | Capture arrays, large pools with high redundancy |
| CustomArray | Electrochemical | 100-200 nt | 8-15% | 1/300-600 bp | 1.5-2× | Mid-size libraries, cost-sensitive projects |
| IDT (Column) | Phosphoramidite | 15-200 nt | <1% | 1/1000-3000 bp | 1× | Small libraries (<100 oligos), primers, high-fidelity |
Note: Dropout rates and error rates are for 200 nt oligos at standard complexity (45-55% GC). Actual performance varies with sequence composition. Data compiled from vendor specifications and published studies (Kosuri & Church 2014; Plesa et al. 2018). Updated November 2024.
Coverage Design Cost Estimation
Budget Planning Framework
Estimate total synthesis costs based on coverage requirements and platform choice:
Array-Based Synthesis (Twist, Agilent)
- • Cost structure: $0.07-0.15 per base (volume-dependent)
- • Typical 10 kb gene project: 63 oligos × 200 nt = 12,600 bases = $880-1,890
- • With 1.5× redundancy: 95 oligos = 19,000 bases = $1,330-2,850
- • Minimum order: Usually 5,000-10,000 oligos (pool capacity)
- • Turnaround: 2-3 weeks
Column Synthesis (IDT, GenScript)
- • Cost structure: $3-8 per oligo (length-dependent, 150-200 nt)
- • Typical 10 kb gene project: 63 oligos × $5 avg = $315
- • With 1.5× redundancy: 95 oligos = $475
- • Purification options: Standard desalting (included), HPLC (+$10-20/oligo), PAGE (+$15-30/oligo)
- • Turnaround: 1-5 days (standard), same-day available
Cost Optimization Strategies
- Reduce oligo length: 150 nt vs 200 nt saves 25% array synthesis cost, but requires more oligos (trade-off).
- Optimize redundancy: Use calculator results to find minimum redundancy that meets quality targets. Each 0.5× reduction in redundancy = 33% cost savings.
- Platform selection: For <100 oligos, column synthesis is cheaper. For >1,000 oligos, array synthesis costs 10-50× less per base. Break-even typically at 300-500 oligos.
- Pool multiple projects: Combine designs to reach array synthesis minimums (5,000+ oligos) and share fixed costs.
- Accept higher error rates: Agilent costs 30-50% less than Twist but requires higher redundancy - calculate total cost including redundancy adjustment.
Complete Oligo Pool Design Workflow
Use these tools in sequence for complete oligo pool design from coverage calculation to synthesis:
Frequently Asked Questions
The optimal overlap depends on your assembly method and synthesis platform:
- Gibson Assembly: 20-40 bp overlap recommended. 30-40 bp is most reliable. Shorter overlaps may fail, longer overlaps waste oligo space. Use Tm Calculator to verify overlap Tm > 48°C for reliable annealing.
- Golden Gate / Type IIS: Overhang length is enzyme-specific: BsaI/BbsI create 4 bp overhangs, SapI creates 3 bp overhangs. Recognition sites add 6-7 bp per side. Minimum functional design typically includes overhang + 10-15 bp flanking for stability. Total oligo = insert + recognition sites (2×6-7 bp) + overhangs (2×3-4 bp).
- PCR-based Assembly: 25-35 bp overlap. Must be long enough for primer annealing (typically Tm > 55°C). Check Secondary Structure to avoid hairpins in overlap regions.
- Ligation: Exact adjacent placement (0 bp spacing) with 5' phosphate modifications. Requires high-fidelity synthesis (column-based preferred).
Platform-specific overlap recommendations (2024-2025): Twist Bioscience (2-5% dropout, high fidelity) works well with 30-35 bp overlap for Gibson assembly; Agilent/CustomArray (15-25% dropout, higher error rate) require 35-45 bp overlap to compensate for synthesis quality. Column synthesis (IDT) can use minimum 20-25 bp due to <1% dropout rate.
The number depends on your screening goals:
- 3-5 gRNAs per gene (typical): Provides redundancy to account for inactive gRNAs. Recommended for genome-wide screens.
- 10+ gRNAs per gene (tiling): For identifying functional domains or essential regions within genes. Provides positional information.
- 1-2 gRNAs per gene (minimal): Only for small-scale or preliminary screens. Risk that some gRNAs won't work.
PAM Site Availability vs. Usable gRNAs: Theoretical calculations based on spacing must be adjusted for:
- • PAM frequency (both strands): SpCas9 (NGG) = 12.5%; SaCas9 (NNGRRT) = 9.4%; Cas12a (TTTV) = 6.25%
- • Design filters remove: Off-targets (20-30%), poor GC content (10-15%), poly-T terminators (5-10%)
- • Final usable yield: High-stringency = 40-60%; Standard = 60-80%; Minimal filtering = 70-85%
Example: For 100 bp spacing theoretical calculation = 10 positions per kb. With SpCas9 PAM density (12.5%) = 1.25 PAM sites on average. After filtering (60% usable) = 0.75 high-quality gRNAs per 100 bp region.
Design workflow: Follow our CRISPR Library Design protocol for PAM identification, off-target analysis, and efficacy scoring. Validate with Batch Sequence QC.
Redundancy requirements vary by application and synthesis method:
- 1x (Single Coverage): Only for column-based synthesis (IDT, GenScript) with <1% dropout. Not suitable for array-based platforms due to dropout rates (2-25%).
- 1.2-1.5x: Optimal for Twist Bioscience (2-5% dropout). High quality enables lower redundancy, reducing costs while maintaining complete coverage.
- 1.5-2x: Recommended for CustomArray (8-15% dropout). Balances cost and reliability for mid-quality array synthesis platforms.
- 2-2.5x: Required minimum for Agilent/SurePrint (15-25% dropout). Higher redundancy compensates for inkjet synthesis variability and ensures complete coverage.
- 3x+: Only for clinical/diagnostic applications requiring guaranteed coverage, or when using lower-quality platforms for critical sequences. Significant cost increase.
Cost vs. Quality: Each increase in redundancy multiplies your oligo count and cost proportionally. Use Error Rate Calculator to determine optimal redundancy for your synthesis platform and Uniformity Estimator to predict dropout impact. Compare synthesis options at Vendor Comparison.
Gaps occur when oligos don't completely cover the target region. Solutions:
- Reduce Spacing: Decrease the gap/spacing between oligos to ensure continuous coverage.
- Increase Redundancy: Higher redundancy will add more oligos and may eliminate gaps.
- Manual Gap Filling: Design specific oligos to cover identified gap regions (use the gap position information from results).
- Adjust Oligo Length: Longer oligos cover more space per oligo, potentially eliminating edge gaps.
Small gaps (<10 bp) at the very end of the target region are often acceptable and can be ignored if they don't affect critical sequences.
Consider these factors for your coverage design:
- • Twist Bioscience: >5,000 oligos; need 150-300 nt high quality (2-5% dropout); gene synthesis, CRISPR libraries; budget $0.09-0.12/base
- • Agilent/CustomArray: >1,000 oligos; can use 120-200 nt; accept 15-25% dropout with 2-2.5× redundancy; capture arrays; budget $0.07-0.09/base
- • Cost advantage: 10-50× cheaper than column synthesis for large pools (>500 oligos)
- • Trade-off: Must design redundancy to compensate for dropout; minimum order quantities apply
- • Small libraries: <100 oligos where per-oligo cost ($3-8) is acceptable
- • High fidelity required: <1% dropout, 1/1000-3000 bp error rate; every oligo must work
- • Fast turnaround: 1-5 days vs 2-3 weeks for arrays; same-day options available
- • Purification options: HPLC, PAGE for critical applications (primers, cloning)
- • 1× coverage sufficient: No redundancy needed due to superior quality
Decision matrix: See our Platform Comparison Table in the guide above for detailed specifications. Break-even point typically at 300-500 oligos depending on length and redundancy requirements.
This calculator provides theoretical estimates assuming:
- • All designed oligos can be successfully synthesized
- • No sequence-dependent synthesis failures
- • Uniform coverage (no preferential amplification or dropout)
- • Perfect spacing (actual designs may need adjustments for sequence constraints)
Real-world adjustments (2024-2025 data):
- • Platform dropout: Twist 2-5%, CustomArray 8-15%, Agilent 15-25% - use redundancy to compensate
- • CRISPR PAM constraints: 12.5% PAM frequency (SpCas9) reduced to 40-80% usable after filtering for off-targets, GC content, efficacy (Doench 2016 criteria)
- • Sequence complexity: High GC (>65%), low GC (<35%), or strong secondary structures (ΔG < -3 kcal/mol) may require redesign - affects 5-15% of theoretical coverage
- • Edge effects: Terminal 10-20 bp at target boundaries may have reduced coverage - design 5-10 bp overhang
Validation workflow: Use Batch Sequence QC for GC/complexity checks, Secondary Structure Predictor for hairpin detection, and follow CRISPR Library Design workflow for comprehensive off-target analysis.
Related Tools
Error Rate Calculator
Calculate expected synthesis quality and full-length product percentage
Batch Sequence QC
Validate your designed sequences for quality issues before synthesis
Pool Uniformity Estimator
Estimate uniformity and dropout rate for your oligo pool
GC Content Analyzer
Analyze GC percentage distribution across your oligo library
Secondary Structure Predictor
Detect hairpins and dimers that may affect assembly efficiency
Tm Calculator
Calculate melting temperatures for PCR primer design and annealing