Sequencing Read-Back Verification — NGS/Nanopore Consensus vs Reference
Align many NGS/Nanopore/Sanger reads to a reference with minimap2, call multi-read consensus variants, and build a corrected consensus sequence.
🌐 Runs on the SeqBench API — also callable via REST & MCP, and in bulk from the batch tools
Check what a real sequencer actually read back from a batch of reads, not just one. Paste (or upload) a reference sequence and a set of raw reads — Sanger, NGS or Nanopore, FASTA or FASTQ, auto-detected — and every read is aligned to the reference with the real minimap2 binary. Each read's cs difference string is parsed into exact substitution/insertion/deletion calls, then compared across reads: a disagreement seen in only one read is reported separately as likely sequencing noise, while a disagreement multiple reads agree on is a consensus-supported variant. The tool goes one step further than a diff list and applies every consensus-supported edit back onto the reference, producing a corrected consensus sequence — what the real construct's sequence appears to actually be. This complements verify_construct and verify_assembly, which check a DESIGN against its own stated inputs; this checks what a real sequencer actually read back from the tube.
0 bp
0 characters pasted — up to 2000 reads / 5,000,000 bp per call.
Reads that must agree on the same edit at a position for it to count as consensus-supported rather than single-read noise.
Paste a reference and its reads to align them with minimap2 and build a consensus.
Reads are aligned to the reference with the real minimap2 binary, and each read's difference string is parsed into exact substitution/insertion/deletion calls. This is a mapping/diff tool, not a variant caller: read-quality (Phred) scores are not used to weight calls, and reads that fail to map at all give no positional information about where they diverge.
How to use the Sequencing Read-Back Verification tool
- 1Paste the claimed/expected reference sequence, and paste raw reads as FASTA or FASTQ (or click Load example for a small synthetic multi-read dataset).
- 2Set the minimum number of reads that must agree on the same edit before it counts as consensus-supported rather than single-read noise.
- 3Review the per-read alignment table, the consensus-variant and single-read-only-position tables, the pass/fail gate, and copy or download the corrected consensus sequence as FASTA.
Frequently asked questions
What counts as a "consensus-supported" variant?
A reference position where at least the chosen minimum number of reads (default 2) agree on the exact same edit (the same substitution, insertion, or deletion) relative to the reference. A position where only a single read disagrees with the reference is reported separately as informational, not gated on, since sequencing error versus a real mutation can't be distinguished from one read alone.
How is the corrected consensus sequence built?
Every consensus-supported edit is applied to the reference, working from the highest reference position down to the lowest, which avoids the classic bug where an earlier insertion or deletion shifts the coordinates of every edit after it. If two different edits at the same position are equally well supported, that position is left as the reference base rather than guessed.
Which read types and formats are supported?
Sanger, NGS (Illumina-style short reads) or Nanopore/PacBio long reads, pasted as FASTA or FASTQ — the format is auto-detected. Up to 2,000 reads or 5,000,000 total bp per call. Alignment runs on the real minimap2 binary, not a re-implementation.
Is this a variant caller with quality-score weighting?
No. It's a mapping/diff tool: read-quality (Phred) scores are not used to weight calls, and reads that fail to map at all give no positional information about where they diverge. Treat consensus support as a count of independent reads agreeing, not a quality-weighted confidence score.
Is my data stored, and can I run this from code?
Your reference and reads are sent to the SeqBench API to run the alignment and are not stored. The same tool, including the gate and the corrected consensus sequence, is available via the REST API and the MCP server.
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