Pairwise sequence aligner

Pairwise alignment answers a simple question: how similar are these two sequences, and where do they differ?

Typical uses in our workflows are checking a Sanger trace (or exported read) against a reference plasmid, amplicon, or gBlock. Comparing two PCR products or mutant constructs to the parent. Aligning a short protein fragment to a full-length sequence (e.g. after translation or domain cloning). Teaching or demonstrating global alignment before moving on to multi-sequence tools. If you have three or more sequences to compare at once, use the multi-sequence aligner instead. This page is only for two sequences.

The aligner uses Needleman–Wunsch global alignment. “Global” means it tries to align the full length of both sequences, inserting gap characters (-) where one sequence is missing residues the other has. That’s what you usually want when both pieces are meant to be the same locus (e.g. read vs reference), not when you’re searching for a small motif inside a huge genome.

Before you click Align, choose either DNA or Protein. In DNA mode the tool will use the following scoring scheme: match +2, mismatch −1, gap −2 (simple scoring, fine for teaching and routine checks). In protein mode it will use BLOSUM62 with gap open −8 (standard for amino acid comparisons). You can read more about BLOSUM62 here.

FASTA headers (lines starting with >) are ignored; only the sequence letters are used. For proteins, selenocysteine (U) is treated as cysteine (C).

The result shows sequence A and B on two lines with a middle row of symbols: | for identical positions, and (for protein) : / . for more or less favourable substitutions. A short summary reports identity and score.

1. Paste Sequence A (often your reference or longer sequence) in the top box.
2. Paste Sequence B (your read, mutant, or second construct) in the bottom box.
3. Select DNA or Protein.
4. Click Align.

Tips that save headaches:

• Strip spaces and line breaks if you copy from a PDF or email; plain A/T/G/C or amino acid letters are fine.
• If the read is much shorter than the reference, you’ll see gaps at the ends — that’s normal for global alignment.
• Very long sequences (many tens of kb) can slow the browser; for huge jobs use EMBOSS needle, BLAST, or similar.
• Use Export (text, RTF, PNG, etc.) if you want a figure or snippet for a notebook or report.

This is a quick visual check, not base-calling software, not a replacement for chromatogram review, and not a database search tool. For finding unknown homology across GenBank, use BLAST or your institute’s pipeline. For aligning many sequences, use our multi-sequence tool or Clustal-style software.

Sequences are processed only in your browser; we don’t upload them to our server for this tool. Still, follow your local rules about confidential or patient-linked data.