ATAC-seq (Assay for Transposase-Accessible Chromatin) uses the hyperactive Tn5 transposase, loaded with sequencing adapters, to cut and tag ("tagment") double-stranded DNA wherever it can physically reach it. A nucleosome — 147 bp of DNA wrapped around a histone octamer — sterically blocks Tn5, so insertions concentrate in nucleosome-free regions (NFRs) such as active promoters and enhancers, while densely packed heterochromatin is largely protected.
Nucleosome repeat length: NRL = 147 bp (core) + linker bp
Fragment-size ladder: L_n ≈ n · NRL, n = 0, 1, 2, 3 …
n=0 (<147 bp) → sub-nucleosomal, from open/NFR DNA
n=1 (~NRL bp) → mono-nucleosomal, spans one nucleosome
n=2 (~2·NRL bp) → di-nucleosomal, spans two nucleosomes
Insertion probability: p(accessible) ≈ 1 − Σ footprint(nucleosome_i)
- Accessible chromatin — the fraction of the 4 kb window rendered as open (teal) beads-on-a-string versus nucleosome-packed (grey) stretches; only accessible DNA accepts a Tn5 landing.
- Linker DNA length — sets the nucleosome repeat length (NRL = 147 + linker), which sets the spacing between the periodicity peaks in the ladder and between the beads on the track.
- Tn5 insertion rate — how many tagmentation attempts fire per second across the whole 4 kb region (independent of what's currently in view); landings that fall on a nucleosome footprint bounce off (red flash) and don't produce a fragment.
- Tn5 : DNA concentration — real ATAC-seq libraries are titrated for enzyme-to-chromatin ratio: too little Tn5 under-digests, too much "over-tagments" and skews the library toward very short fragments by cutting each accessible stretch more than once. Raising this slider biases successful cuts toward their nearest neighbouring accessible site instead of one further down the fibre.
- Track zoom / drag — zooms the linear track and lets you drag to pan across the full 4 kb region; the reaction itself always runs across the whole region regardless of what's on screen.
Real-world relevance: the growing bands on the virtual gel panel — a decaying series of bright rows at multiples of the nucleosome repeat length — is a schematic of the standard ATAC-seq QC trace used to confirm a library actually captured nucleosome positioning before it is used for peak calling or, as in the source article, integrated with scRNA-seq and spatial data into a single-cell multi-omics atlas.