Files & formats

Sequencing read-back verification

Align raw Sanger or NGS reads (FASTA or FASTQ) back onto a claimed reference sequence using minimap2, and report per-read mapping identity plus exact variant positions (substitutions/insertions/deletions), with a consensus view across reads and a corrected consensus sequence (the reference with every consensus-supported edit applied). Each alignment also reports how much of the READ was used (queryCoveragePct/clippedBases), since identity is measured over the aligned portion only and a partially-used read would otherwise score perfectly. Set circular: true for a plasmid so reads crossing the reference's arbitrary linear start are aligned through the join rather than cut short at it. Also calls STRUCTURAL variants from split alignments — a large deletion, tandem duplication, inversion or backbone rearrangement never appears as a run of mismatches, only as one read aligning at several distant reference positions, so per-base calling reports a perfect clone — and returns a coverage depth profile with the regions no read reached at all, since "never read" is not "correct". On a circular reference one junction cannot always tell an event of length d from one of length referenceLength − d the other way round; where the read's own blocks and the coverage profile settle it they do, and where they do not the call carries an alternateInterpretation with the other reading rather than presenting one as a finding. Set platform (nanopore/pacbio/illumina/sanger) to pick minimap2's preset; the preset used is reported back. Complements verify_construct/verify_assembly: those re-derive what a design SHOULD produce from its own stated inputs; this checks what a real sequencer actually read back.

post/sequencing_readback_verify

Request

  • Base URL: https://seqbench.com/api/v1
  • URL: https://seqbench.com/api/v1/sequencing_readback_verify
  • Auth: none required

Request body

referencestring required

The claimed/expected reference — bare bases, FASTA, or a whole GenBank record. Pass the GenBank record (your own map) to get consequences: every consensus edit is then placed against the features, so the answer reads "silent in AmpR" or "premature STOP at codon 41 of your CDS" instead of only a position and a base. Bare bases still work and simply return no consequences.

readsstring required

Raw reads in FASTA or FASTQ format (auto-detected). Up to 2000 reads / 5,000,000 total bp per call.

minSupportingReadsinteger

Minimum number of reads agreeing on a variant position for it to count as a consensus (candidate real) variant rather than single-read noise.

circularboolean

Treat the reference as a circular molecule (plasmid). Reads that straddle its arbitrary linear start are then aligned right through the join instead of being cut short there, so variants in the part that would otherwise be clipped away are actually called. Turn this on for whole-plasmid data — the reads begin wherever the molecule was cut, so most of them cross the join. Reads longer than the reference still get clipped. It also governs structural-variant calling: on a circle an origin-crossing read is contiguous, and only this flag lets it be told apart from a whole-reference duplication.

platform'auto' | 'nanopore' | 'pacbio' | 'illumina' | 'sanger'

Read type, which selects minimap2's alignment preset. "auto" (the default) passes no preset and uses minimap2's own defaults — whose parameters happen to be map-ont's, so nanopore data is already handled; setting "nanopore" makes that explicit and pins it. "pacbio" uses map-hifi and "illumina" uses sr, both of which materially change the result: under the ONT defaults gaps are cheap, so on accurate reads a cluster of mismatches can be reported as a spurious indel. "sanger" uses map-hifi with the minimum alignment score returned to 80, because map-hifi's own floor of 200 was chosen for 10-25 kb reads and would leave a short or partly-failed Sanger read unmapped. The preset actually used is reported back as minimap2Preset/alignerArgs so a run can be reproduced. Names are matched ignoring case and punctuation, and the obvious aliases resolve ("ONT" and "map-ont" to nanopore, "HiFi" to pacbio, "sr" to illumina); anything unrecognized is rejected rather than silently treated as "auto".

minSvSupportinteger

Distinct reads that must agree on the same breakpoint before a structural variant (large deletion, duplication, inversion, insertion) is reported. Default 3 — stricter than minSupportingReads on purpose: a single split read is a library-prep chimera or a mapping artifact far more often than it is biology, and chimeras are common enough that two agreeing by chance is likelier than two matching base errors.

minSvLengthinteger

Smallest structural variant to report, in bp (default 50). Below this the per-base variant caller already reports the event from inside a single alignment, and on nanopore data the range is mostly the error profile.

breakpointToleranceinteger

How far apart two reads' breakpoints may sit and still count as the same event, in bp (default 50). Long-read breakpoints are fuzzy, and microhomology at a real junction moves the split legitimately.

Response

Tool ran successfully.

okunknown required
toolstring required

The tool slug that ran.

resultobject required

Tool-specific output object. Its shape depends on the tool — call GET /{tool} or see the docs for each tool's fields.

Example response

{
  "provenance": {
    "apiVersion": "1.1.0"
  }
}

Changes

No recorded changes to this operation across all 1 revision of this API.