Verify vs Reference

Compare a sequencing read against the sequence you designed, list every difference in the reference's own numbering, classify each one against a CDS, and mark them on the reference itself.

What it doesCopy Anchor Link

A read that "looks fine" is not the same as a read that matches what you meant to build. This section answers the second question: it aligns the read to a reference sequence and reports every position where the two differ, numbered against the reference rather than against the read.

That distinction is the whole point. A read has its own coordinates, which start wherever the primer landed and shift by one at every insertion or deletion; the reference's coordinates are the ones your construct map, your primers and your collaborators all use. A difference reported at "read base 412" is almost useless. The same difference reported at "reference base 1,187, in the middle of the CDS, changing Ala to Thr" is a result.

With a coding sequence available, each difference is also classified: silent, missense, nonsense, or a frameshift. That classification is made in the reference's frame, so an indel upstream correctly makes everything after it a frameshift rather than a run of independent substitutions.

When you'd use thisCopy Anchor Link

• After cloning, to check the insert you sequenced is the insert you designed.
• After mutagenesis, to confirm the change you wanted is there and nothing else came with it.
• When a colony sequences "almost right" and you need to know whether the differences are real or are the read's degraded ends.
• Before you tell anyone the construct is correct.

What this computesCopy Anchor Link

• A count of differences between the active read — edits and trims applied — and a reference
• A row per difference in the reference's own numbering: reference base, read base, position, and its consequence where a coding sequence is available
• The Reference canvas view, drawing the read's own peaks under the reference column by column, so each difference can be judged from the trace rather than from a letter
• Annotate reference, writing every difference onto the reference as a real feature so it travels with the record rather than living only in this panel

Where it livesCopy Anchor Link

The Verify vs reference section of the Chromatogram panel, acting on the active trace tab. Its header carries a detach control that floats the whole section above whatever else you are looking at.

Detach it before you annotate. Annotate reference writes onto the reference, so it is enabled only while the reference is the focused tab — and focusing the reference replaces the right rail with that sequence's own inspector. Floating the section is what keeps the control on screen while you switch, and it is the intended path rather than a workaround.

Inputs and outputsCopy Anchor Link

In: the active read, with its edits and trims applied, plus a reference — an open tab, a file in the project, or an upload.

Out:

• A count of differences, and a row per difference in the reference's numbering: reference base, read base, position, and its consequence when a CDS is available.
• The Reference canvas view, which draws the read's own peaks under the reference column by column, so each difference can be judged from the trace rather than from a letter.
• Annotate reference — every difference written onto the reference as a real feature, so it travels with the sequence and appears in an export.
• Report (TSV) — the whole table, for a notebook or a supplement.

What it refuses, and what it says:

• No reference chosen — the section says how to pick one and the Reference view says the same rather than drawing an empty frame.
• A read that will not align to the reference — you are told to check the template and the strand, since a reverse read compared to a forward reference is the usual cause.
• Annotating while the reference is not focused — the control is disabled and its tooltip names the tab you need to focus.

Reading the resultCopy Anchor Link

Two reads of the same clone rarely disagree with a reference in the same places, and that is diagnostic in itself:

• Differences clustered at one end are almost always the read degrading, not the template differing. Trim and re-run.
• Differences spread evenly at low density usually mean you are comparing against the wrong template, or the right template on the wrong strand.
• One or two differences in the good middle of the read, on clean single peaks, are real, and are what this tool exists to find.

Where a difference sits on a double peak rather than a single one, the template is mixed at that position — a heterozygote or an incompletely edited population — and the base caller had to pick one letter. The Reference view is where you see that; the table cannot show it.

RelatedCopy Anchor Link

• Chromatogram — the editor this section belongs to, including the Contig view for merging a forward and reverse read before verifying.
• Read Alignment — screening a read for vector, and aligning reads to each other rather than to a reference.