---
title: "How to Analyze Hotspots and Synergies"
slug: "how-to-analyze-hotspots-and-synergies"
tags: ["Discngine Ideation Analytics", "Peptides"]
updated: 2026-09-24T08:48:57Z
published: 2026-09-24T08:48:57Z
canonical: "docs.discngine.com/how-to-analyze-hotspots-and-synergies"
---

> ## Documentation Index
> Fetch the complete documentation index at: https://docs.discngine.com/llms.txt
> Use this file to discover all available pages before exploring further.

# How to Analyze Hotspots and Synergies

**Hotspot analysis** scores how much each position (and optionally bridges, chemical side chains, and regions) impacts a chosen endpoint. **Synergy analysis** goes further and finds combinations of positions that act *together*. Both are configured on the **Hotspot** tab of the SAR Report settings drawer. This guide covers every option in detail.

              Learn more

              

For how the scores are computed and how to avoid over-reading them — why some element always reads 100%, what a `N/A` synergy means, and why strength is not activity — see [Understanding Hotspots and Synergies](/public/docs/explanation-understanding-hotspots-and-synergies).

## How hotspot analysis works

Hotspot analysis identifies the positions with the highest structure-activity relationship (SAR) impact by measuring how much a property varies across the different residues (**variants**) seen at each position.

For each position, the algorithm:

1. **Groups** the peptides by their variant at that position.
2. Computes a **baseline** using the selected aggregation method (average, median, or the reference peptide's value).
3. Calculates the **range of variant deviations** from that baseline.
4. Computes a **delta** for each variant (variant's displayed value − baseline). The variant's displayed value follows the endpoint's aggregation (max, min, average, median; histogram uses the average, boxplot the median) — see [How to Explore the Peptide Viewer](/public/docs/how-to-explore-peptide-viewer).
5. **Normalizes** the impact to 0–100% against the largest range in the dataset.

For a **region** (a group of positions), the algorithm first builds a structural **fingerprint** combining every monomer, chemical object, and bridge inside the region, groups peptides by that fingerprint, and then scores impact with the same aggregation and normalization as a single position.

A higher impact means residue substitutions at that spot move the property more — making it a key target for optimization.

## Enable hotspot analysis

Open **Settings → Hotspot** and turn on **Enable Hotspot Analysis**. Impact bars and significance badges immediately appear on the canvas.

![The Hotspot tab with analysis enabled, the configuration panel, and impact bars with Hot/Warm/Cold coloring on the canvas](https://discngine.github.io/ideation/Images/how-to-analyze-hotspots-and-synergies/enable-hotspot-analysis.png)

## Configure the analysis

### Endpoint

The numeric endpoint to score (for example `pIC50`). Only numeric endpoints are selectable.

### Baseline Aggregation Method

How the baseline that variants are compared against is computed. This affects both the gallery deltas and the impact scores.

| Method | Baseline |
| --- | --- |
| **Average value of all peptides** | The mean of all peptide values at the position. |
| **Median value of all peptides** | The median of all peptide values at the position. |
| **Reference peptide value** | The value of the reference peptide (set on the Data tab). |

### Include Elements

Choose which element types get hotspot scoring, coloring, bars, and badges:

| Toggle | Applies hotspot analysis to… |
| --- | --- |
| **Include Monomers** | Amino-acid positions. |
| **Include Chemical Side Chains** | Side chemical chains, each scored as a whole chain. |
| **Include Bridges** | Disulfide and lactam bridges. |
| **Include Regions** | Grouped regions (scored via the region fingerprint). |

### Thresholds

A dual-handle slider (0–100%) that sets the **Warm** and **Hot** cut-offs used to classify each element. The defaults are **Warm 50%** and **Hot 70%**:

- **Hot** — impact ≥ the Hot threshold (high impact).
- **Warm** — Warm threshold ≤ impact < Hot threshold (moderate impact).
- **Cold** — impact < the Warm threshold (low impact).

## Read the results

Each included element on the canvas shows:

| Cue | Meaning |
| --- | --- |
| **Impact bar** | A horizontal bar filled to the element's relative impact (0–100%); the fill color reflects strength. Shows the quantitative *magnitude*. |
| **Significance badge** | 🔴 **Hot**, 🟠 **Warm**, 🟢 **Cold** — at the default thresholds, ≥ 70%, 50–70% and < 50% respectively. Shows the qualitative *classification*. |
| **Gallery deltas** | In galleries: green **+** = higher activity, red **−** = lower activity, grey `0.0` = no change. Delta = variant value − baseline. |

![The synergy detection settings and the hotspot rendering legend (impact bar, Hot/Warm/Cold badges, and delta values)](https://discngine.github.io/ideation/Images/how-to-analyze-hotspots-and-synergies/read-the-results.png)

The **Display options** section controls how results are drawn:

| Option | Effect |
| --- | --- |
| **Color Scale** | The impact color ramp: **Heat Map (Red-Yellow-Green)**, **Viridis (Purple-Green-Yellow)**, or **Red-Blue**. |
| **Show Impact Bars** | Show/hide the 0–100% impact bars. |
| **Show Significance Badges** | Show/hide the Hot/Warm/Cold dots. |
| **Show Gallery Deltas** | Show/hide the per-variant delta values in galleries. |
| **Highlight Only Significant Positions** | When on, only **Hot** and **Warm** elements are styled; Cold elements stay plain, so the impactful spots stand out. |

With hotspot enabled, the Display tab also gains a **Hotspot** option under **Color monomers by**.

## How synergy detection works

Single-position hotspot analysis scores each position on its own, so it cannot see positions that only matter **in combination**. Synergy detection fills that gap:

1. Peptides are grouped by the **combination** of residues at a set of 2–3 positions (or a contiguous region).
2. An **additive prediction** is built for each combination: the overall mean plus each position's *individual* effect.
3. The **synergy** is how far the combination's observed activity deviates from that additive prediction — a consistent gap is the interaction signal.

Interaction types:

- **Synergistic** — together *more* active than the sum of the individual effects (the positions reinforce each other).
- **Antagonistic** — together *less* active than expected (the positions interfere).
- **Neutral** — activity matches the additive prediction.

Each detected synergy is drawn as a **region** on the viewer (for example "Synergy 2 + 5").

## Detect multi-position synergies

In the **Synergies** section, turn on **Detect multi-position synergies**.

![The Synergies section with multi-position synergy detection enabled and the detection parameters](https://discngine.github.io/ideation/Images/how-to-analyze-hotspots-and-synergies/detect-multi-position-synergies.png)

### Detection options

| Option | What it controls |
| --- | --- |
| **Combination order** (default *Pairs and triples*) | How positions are grouped: **Pairs only** (2), **Pairs and triples** (2–3), **Triples only** (3) — these may be non-adjacent — or **Contiguous regions**, which scans adjacent stretches of monomers (useful for motif-like synergies longer than 3). |
| **Region length range** (regions mode only, default 2–6) | The shortest and longest contiguous run of monomers to scan. Longer regions need more peptides sharing the same motif, so they are often dropped for low support. |
| **Min. peptides per combination** (default 3) | The minimum number of peptides that must share the same variant combination for it to count. Higher = more reliable but needs more data; combinations below it are ignored. |
| **Ignore gap combinations** (default on) | Exclude combinations that include an empty position (a gap / "None"), so synergies are scored only on actual residue combinations. |

## Filter and sort the synergies

| Control | What it does |
| --- | --- |
| **Min. synergy strength (% of strongest)** (default 70%) | Hides synergies below a minimum **interaction strength**. Each set's raw score is the support-weighted RMS of its combinations' residuals (observed − predicted), scaled against the strongest set *of the same size* (pairs, triples, and regions each scaled to their own strongest). 0% lists everything; 100% keeps only the strongest. This is interaction strength, **not** activity. |
| **Filter by activity impact** | **Any activity** (all), **Improves activity** (only sets with a combination that is both synergistic *and* more active than the baseline), or **No activity gain** (sets that interact but not toward a more active combination). |
| **Sort by** | **Strength** (relative magnitude), **Most synergistic** (largest share of synergistic combinations), **Most antagonistic** (largest share of antagonistic combinations), or **Activity impact** (best synergistic + active combination first). |

Each detected synergy is also drawn as a **region** on the viewer, connecting its positions (for example *Synergy 10 + 30*). Clicking one opens a synergy region gallery:

![Synergy regions drawn on the viewer &#x2014; Synergy 10 + 30 with its connectors and a synergy region gallery, over the hotspot impact bars](https://discngine.github.io/ideation/Images/how-to-analyze-hotspots-and-synergies/filter-and-sort-the-synergies-01.png)

The synergy list shows a count (**"N synergies found"**) and one row per synergy:

![The synergy results list showing 97 synergies, each with a checkbox, positions, a synergistic/antagonistic direction bar, strength percentage, and its best combination](https://discngine.github.io/ideation/Images/how-to-analyze-hotspots-and-synergies/filter-and-sort-the-synergies-02.png)

Each row has a **checkbox** (show the synergy as a region on the viewer), the **positions**, a **direction bar** (green synergistic / grey neutral / red antagonistic), the **strength %**, and the **best combination** — its residues, the **Activity** and **Synergy** deltas, and the supporting peptide count *n*. Until you tick or untick a row yourself, only the **three sets at the top of the list**, in its current sort and filter, are drawn on the viewer. Use **Check all** / **Uncheck all** to toggle every row currently listed, and **Explore** to open the combinations table. Deleting a synergy region from its right-click menu on the canvas unticks its row.

              If no synergy is found

              

If nothing meets the settings you will see **"No synergy found"** — lower the **Min. synergy strength** or **Min. peptides per combination**, change the activity filter, or add more peptides.

## Explore synergy combinations

Click **Explore** to open the **Explore Synergy Combinations** dialog — a sortable table of every combination across the listed synergies. Each row shows the **Positions**, the residue **Combination**, its **Strength**, the **Synergy Δ** and **Activity Δ**, the supporting peptide count **n**, and a **box-plot per property**. Use **Group by positions** and **Synergistic & active only** to focus the table, and click a column header to sort.

![The Explore Synergy Combinations dialog: a sortable table of combinations with Synergy and Activity deltas and per-property box-plots](https://discngine.github.io/ideation/Images/how-to-analyze-hotspots-and-synergies/explore-synergy-combinations.png)

## Next steps

- [Understanding Hotspots and Synergies](/public/docs/explanation-understanding-hotspots-and-synergies) — how the scores are computed and how to read them
- [How to Cluster Peptides](/public/docs/how-to-cluster-peptides)
- [Understanding Peptides in Ideation](/public/docs/explanation-understanding-peptides)