Sanger Read vs Reference — Verify a Clone Against a Template
Align a Sanger read to a reference and get a pass / needs-review verification report.
🔒 Local processing — pasted sequences are not uploaded
Confirm that a Sanger read matches the sequence you expected. Paste a read in FASTA (or upload an .ab1 trace and its base calls are extracted for you) together with the reference template; the tool aligns them end-to-end with a global Needleman–Wunsch alignment and produces a verification report — percent identity, every substitution, insertion and deletion with reference coordinates and HGVS-style notation, a colored side-by-side diff, and a clear pass / needs-review verdict. It is the fast way to check a cloned construct or a mutagenesis product against its intended sequence before moving on.
0 bp
0 bp
Paste (or upload) a Sanger read and a reference sequence to verify the read against the expected template.
The read and reference are aligned end-to-end with a global (Needleman-Wunsch) alignment; every mismatch, insertion and deletion is called against the reference with a 1-based coordinate and HGVS-style g. notation. The verdict is a triage aid — a read is marked PASS at ≥99% identity with no indels, otherwise NEEDS REVIEW. Always confirm called differences by eye on the chromatogram (in the Sanger Trace Viewer), since low-quality base calls near the read ends are the most common source of spurious mismatches.
How to use the Sanger vs Reference tool
- 1Paste the reference (expected) sequence.
- 2Paste the Sanger read as FASTA, or upload an .ab1 / .abi trace to pull in its base calls.
- 3Read the verdict, percent identity and the mismatch/indel table, and inspect the colored diff at each difference.
Frequently asked questions
- How do I provide the read — paste or file?
- Either. Paste the read as FASTA or raw text, or upload the .ab1 / .abi trace and the tool extracts its base calls for you. The reference is always pasted. The read is decoded locally in your browser.
- How is the pass / needs-review verdict decided?
- The read and reference are aligned globally, and the read is marked PASS at 99% or higher identity with no insertions or deletions; otherwise it is flagged NEEDS REVIEW. It is a triage aid — always confirm called differences by eye on the chromatogram, since low-quality base calls near the read ends are the most common source of spurious mismatches.
- What differences does the report list?
- Every substitution, insertion and deletion between the read and the reference, each with a 1-based reference coordinate and HGVS-style g. notation (e.g. g.54G>A, g.54del, g.54_55ins), plus counts of mismatches, insertions and deletions and the overall percent identity.
- How are the two sequences aligned?
- With a global (Needleman–Wunsch) alignment, so the whole read is compared end-to-end against the reference. That suits a single Sanger read versus its expected template rather than searching a large genome.