Base composition and GC content

Base composition is the simplest thing you can measure about a sequence: how many of each base it contains. From those counts come GC content, AT content, and a quick read on sequence quality.

The counts

Tally A, C, G, T and N across the sequence. From the totals:

GC% = (G + C) ÷ length × 100 · AT% = (A + T) ÷ length × 100

For ATGCGC (6 nt): A 1, T 1, G 2, C 2, N 0. GC% = 4 ÷ 6 × 100 =66.7%; AT% = 2 ÷ 6 × 100 = 33.3%. The two add to 100% only when there are no ambiguous bases.

Where N fits

An N is a position the basecaller couldn’t resolve. It counts toward the length but is neither GC nor AT, so a sequence with many Ns shows GC% + AT% below 100% — a useful flag that a stretch is low quality or masked.

Why GC content matters

GC-rich and AT-rich regions behave differently in the lab. G≡C pairs are more stable than A=T pairs, so GC content shifts melting temperature, affects primer and probe design, and can cause uneven coverage — GC-extreme regions amplify and sequence less efficiently. A composition check is often the first look at why a region is hard.

A quick profile

Where the GC content tool gives just the percentage, a full base-composition breakdown shows the exact A/C/G/T/N counts alongside both GC% and AT% — handy for confirming a synthetic construct, checking strand balance, or spotting an unexpected skew before you design primers against a region.