Base composition per cycle

What fraction of the pool reads A, C, G and T at each index cycle, with the Shannon entropy of every cycle beneath it.

What it doesCopy Anchor Link

Each column is one index cycle — the first base of every barcode, then the second, and so on. The column is stacked to 100% and split by base, so its four segments are the fraction of the pool reading A, C, G and T at that cycle. Hovering a column gives that cycle's entropy in bits, and the note under the plot lists the entropy of every cycle in order.

This is the sequencer's-eye view of a barcode set. The instrument does not read barcodes; it reads cycles, across the whole pool at once. A cycle where every sample reads the same base is a cycle with no information in it, and on some chemistries it is worse than useless — see the channel-occupancy plot for what happens when that shared base is a dark one.

When you'd use thisCopy Anchor Link

• Before committing a pool to a run, as the second check after colour balance. A low-entropy cycle is visible here in a way it is not in a table of sequences.
• When your set is a subset of a plate. Vendor index sets are balanced as a whole; twelve wells picked from them frequently are not, and this plot is where that shows up.
• When base calling or cluster registration was poor on a previous run with the same indexes.
• While hand-editing a set — the plot redraws per edit, so you can watch a flat cycle open up.

What this computesCopy Anchor Link

• Per-cycle base fractions — the fraction of the pool reading A, C, G and T at each index cycle, a straight count over the pool divided by the set size, with no weighting by expected library yield because that is not something a barcode set knows
• Per-cycle Shannon entropy in bits, H = −Σ p log₂ p, so 2 bits is the maximum (all four bases equally represented), 1 bit is a clean two-base split and 0 bits is every sample reading the same base. The values are printed on that scale and say "of 2 bits", because an entropy number with no stated base and no stated maximum is unreadable

What it refuses: a mixed-length set. There is no shared notion of "cycle 3" across lengths, so the analysis records a note naming the lengths it found and the section does not render, rather than drawing something misleading.

What entropy does not tell you: it is base-blind. Two cycles can both read 1 bit while one is a healthy A/C split and the other an A/G split with G dark on your instrument. Entropy measures spread, not signal — per-cycle channel occupancy is the plot that knows about dyes.

Where it livesCopy Anchor Link

The Analysis tab of the Barcode & Index Design panel, below the GC distribution, with the per-cycle entropy printed beneath the plot. The section header carries the detach control.

Inputs and outputsCopy Anchor Link

In: the panel's working set, which must be equal-length for this plot to exist. A mixed-length set has no shared notion of "cycle 3", so instead of drawing something misleading the analysis records a note naming the lengths it found, and the section does not render.

Out: per-cycle base fractions summing to 1, and per-cycle Shannon entropy in bits.

ControlsCopy Anchor Link

The scienceCopy Anchor Link

The fractions are a straight count over the pool at each position, divided by the set size — no weighting by expected library yield, because that is not something the barcode set knows.

Entropy is Shannon entropy in bits over the observed base distribution at that cycle:

H = −Σ p log₂ p

so 2 bits is the maximum, reached when all four bases are equally represented; 1 bit is a clean two-base split; 0 bits is every sample reading the same base. The values printed under the plot are on that scale, which is why the note says "of 2 bits" — an entropy number with no stated base and no stated maximum is unreadable.

Reading the scale in practice: at 2 bits the cycle is ideal. Around 1.5 bits it is comfortable. Below 1 bit one base dominates, and you should check that cycle against the chemistry's channel map. At 0 bits the cycle carries no information at all, and if that base is dark on your instrument the pool cannot be base-called at that cycle.

LimitationsCopy Anchor Link

• Equal lengths only, for the reason above.
• It weights every barcode equally. A real pool is loaded at deliberately unequal molarity, and a cycle that looks balanced across the set can be unbalanced across the actual flow cell.
• Entropy is base-blind. Two cycles can both read 1 bit while one is a healthy A/C split and the other an A/G split with G dark on your instrument. Entropy measures spread, not signal — the channel-occupancy plot is the one that knows about dyes.

RelatedCopy Anchor Link

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