Tier Classification

How SPACER classifies guide RNAs into quality tiers based on their assay score.

Overview

After computing the assay score (0.0–1.0) for each candidate guide, SPACER assigns it to one of four quality tiers via SpacerTier::from_assay_score(). Tiers provide a quick, actionable classification that helps researchers prioritize guides for experimental validation without needing to interpret raw scores.

Tier Thresholds

Tier	Assay Score	Label	Color	Meaning
Excellent	≥ 0.80	Tier 1	Green	Top-quality guides with strong scores across all evaluated criteria. Recommended as primary candidates for assay development.
Good	0.60–0.79	Tier 2	Blue	Solid guides with favorable properties. Suitable for experimental validation, especially when Excellent-tier candidates are limited.
Fair	0.40–0.59	Tier 3	Yellow	Acceptable guides with one or more moderate weaknesses. Consider these when higher-tier options are scarce or when targeting a specific region.
Poor	< 0.40	Tier 4	Red	Low-quality guides with significant concerns. Not recommended for use without compelling biological reasons and additional validation.

Tier Assignment Logic

Tier assignment is a simple threshold-based classification applied after the assay score is calculated. The canonical implementation is SpacerTier::from_assay_score(), which multiplies the 0.0–1.0 score by 100 and applies integer thresholds:

The assay score (0.0–1.0) is scaled to 0–100 and compared against tier boundaries
Boundary values are inclusive: a score of exactly 0.80 is Excellent, 0.60 is Good, and 0.40 is Fair
Quality flags do not affect tier assignment — a guide with flags can still be Excellent if its assay score is high enough

Info

Quality flags are reported alongside the tier but do not modify it. A guide with an assay score of 0.85 and a POLY_T flag is still classified as Excellent-tier, but the flag serves as a warning that researchers should inspect the specific concern before proceeding.

Typical Tier Distributions

The proportion of guides in each tier varies depending on the target sequence, enzyme configuration, and enabled scoring components. Typical distributions for well-characterized viral targets:

Tier	Typical %	Notes
Excellent	5–15%	Only the best candidates; more common with Cas13 + ML activity
Good	15–30%	The primary selection pool for most users
Fair	25–40%	Large pool; useful for region-constrained designs
Poor	20–50%	Often guides with extreme GC or homopolymer issues

Practical Selection Guidance

Tiers are designed to support a practical experimental workflow:

Start with Excellent: Select 3–5 Excellent-tier guides as primary candidates for your assay
Supplement with Good: If Excellent-tier guides do not cover your desired target regions, add Good-tier guides to fill gaps
Use Fair for constrained designs: When you must target a specific conserved region and no higher-tier guides are available, Fair-tier guides are acceptable with additional validation
Avoid Poor unless necessary: Poor-tier guides should only be used when no alternatives exist and the biological context demands that specific region

Tip

For SHERLOCK/DETECTR diagnostic assays, aim to validate at least 3 guides from the Excellent or Good tiers. Testing multiple guides increases your chances of finding one with optimal in vitro performance, since computational scoring cannot capture all factors affecting real-world activity.

Overview

Tier Thresholds

Tier	Assay Score	Label	Color	Meaning
Excellent	≥ 0.80	Tier 1	Green	Top-quality guides with strong scores across all evaluated criteria. Recommended as primary candidates for assay development.
Good	0.60–0.79	Tier 2	Blue	Solid guides with favorable properties. Suitable for experimental validation, especially when Excellent-tier candidates are limited.
Fair	0.40–0.59	Tier 3	Yellow	Acceptable guides with one or more moderate weaknesses. Consider these when higher-tier options are scarce or when targeting a specific region.
Poor	< 0.40	Tier 4	Red	Low-quality guides with significant concerns. Not recommended for use without compelling biological reasons and additional validation.

Tier Assignment Logic

The assay score (0.0–1.0) is scaled to 0–100 and compared against tier boundaries

Boundary values are inclusive: a score of exactly 0.80 is Excellent, 0.60 is Good, and 0.40 is Fair

Quality flags do not affect tier assignment — a guide with flags can still be Excellent if its assay score is high enough

Info

Typical Tier Distributions

The proportion of guides in each tier varies depending on the target sequence, enzyme configuration, and enabled scoring components. Typical distributions for well-characterized viral targets:

Tier	Typical %	Notes
Excellent	5–15%	Only the best candidates; more common with Cas13 + ML activity
Good	15–30%	The primary selection pool for most users
Fair	25–40%	Large pool; useful for region-constrained designs
Poor	20–50%	Often guides with extreme GC or homopolymer issues

Practical Selection Guidance

Tiers are designed to support a practical experimental workflow:

Start with Excellent: Select 3–5 Excellent-tier guides as primary candidates for your assay

Supplement with Good: If Excellent-tier guides do not cover your desired target regions, add Good-tier guides to fill gaps

Use Fair for constrained designs: When you must target a specific conserved region and no higher-tier guides are available, Fair-tier guides are acceptable with additional validation

Avoid Poor unless necessary: Poor-tier guides should only be used when no alternatives exist and the biological context demands that specific region

Tip