Focal Amplification and ecDNA Usage Guide

Overview

A depth caller reports that an oncogene is amplified and at what amplitude, but not the structure. Focal amplifications come in distinct architectures: extrachromosomal DNA (ecDNA, circular and episomal), breakage-fusion-bridge cycles, homogeneously staining regions, and simple linear amplification. The architecture determines the biology: ecDNA lacks a centromere, segregates unequally, and can surge in copy number under selection, making it a structural basis for therapy resistance. Resolving architecture requires the breakpoint graph, which is what AmpliconArchitect and the AmpliconSuite pipeline reconstruct. This skill covers seed selection, graph reconstruction, and ecDNA classification.

Prerequisites

# AmpliconSuite-pipeline bundles AmpliconArchitect and AmpliconClassifier
conda install -c bioconda ampliconsuite          # or install from GitHub
conda install -c bioconda cnvkit samtools        # CNV seeding
# Configure $AA_DATA_REPO (reference download) and a free academic Mosek license

Inputs: a whole-genome sequencing BAM (AmpliconArchitect is designed for WGS, not panels/WES); copy-number seeds (high-CN focal regions, ideally from a vetted CNVkit callset); a genome build matching the BAM.

Quick Start

Tell the AI agent what to do: - "Reconstruct the architecture of this focal MYC amplification from WGS" - "Determine whether this high-copy EGFR amplification is ecDNA or a chromosomal HSR" - "Run the AmpliconSuite pipeline end-to-end on this tumor BAM" - "Select copy-number seeds for AmpliconArchitect from my CNVkit calls" - "Explain why a depth caller cannot tell me whether an amplification is ecDNA"

Example Prompts

Architecture reconstruction

"Run AmpliconSuite end-to-end on this tumor WGS BAM in GRCh38 and report which amplicons AmpliconClassifier labels as ecDNA versus BFB versus HSR."

"Seed AmpliconArchitect from my vetted CNVkit focal calls and reconstruct the breakpoint graph for the chr8 amplicon."

Interpretation

"This EGFR amplicon has copy number around 40 and spans three non-contiguous segments. Assess whether this pattern is consistent with ecDNA and what would confirm it."

"AmpliconClassifier returned 'unknown' for a clearly amplified locus. Explain the short-read resolution limit and what orthogonal assay to use."

Scope

"My depth caller says this region is amplified. Explain why that does not establish ecDNA and what breakpoint-graph evidence is required."

What the Agent Will Do

1. Confirm the input is adequate-coverage whole-genome sequencing
2. Build or vet copy-number seeds: focal, high-CN regions only
3. Run AmpliconArchitect to reconstruct the breakpoint graph and optimize balanced flow
4. Classify each amplicon with AmpliconClassifier (ecDNA / BFB / HSR / linear)
5. Cross-check ecDNA calls against the closed-cycle graph signature
6. Recommend orthogonal confirmation (FISH, single-cell, optical mapping) for high-stakes calls

Tips

• Depth establishes that a region is amplified and how much; it never establishes the architecture, since ecDNA and a chromosomal HSR look identical in a depth profile.
• AmpliconArchitect reconstructs the regions it is seeded with; garbage seeds (noisy or arm-level) produce garbage amplicons.
• An ecDNA call requires a closed-cycle breakpoint graph plus an AmpliconClassifier label, ideally with orthogonal confirmation.
• AmpliconArchitect needs whole-genome sequencing and adequate coverage; it is not for panels or WES.
• Set the genome build explicitly; AmpliconArchitect was historically hg19-centric.
• Complex amplicons exceed short-read resolution; escalate to optical mapping or long reads when the graph is fragmented.

Related Skills

• copy-number/cnvkit-analysis - Generates copy-number seeds for amplicon reconstruction
• copy-number/recurrent-cnv - Cohort-level recurrent focal amplification
• copy-number/allele-specific-copy-number - Absolute copy number of amplified loci
• copy-number/cnv-annotation - Oncogene annotation of amplified regions
• copy-number/subclonal-copy-number - Subclonal dynamics of ecDNA copy number
• long-read-sequencing/structural-variants - Long-read resolution of complex amplicons