The technology
One probe per target. One consensus call per molecule.
LockSeq captures each site with a single padlock probe and tags every captured molecule before amplification. Every consensus read traces back to one original molecule, which is how we tell random PCR and sequencing errors apart from real variants.
Built on padlock-probe chemistry from Prof. Mats Nilsson's lab at Stockholm University and SciLifeLab, where Countagen's founders developed LockSeq.
UMI consensus calling
How UMI consensus calling removes stochastic errors
A single sequencing error mistaken for an off‑target edit can stall a whole program.
Every captured molecule gets its UMI before any amplification. After sequencing, reads that share a UMI are collapsed into one consensus call (Hiatt et al. 2013).
A true variant shows up in every read from that molecule. A random PCR or sequencing error shows up in only some, so the consensus drops it. That is what lets LockSeq call variants down to 0.1% VAF with confidence. Each site’s limit is set by its own coverage.
What consensus does to the data
In standard NGS reads, a true variant (violet) can hide in the background of random PCR and sequencing errors (grey). Consensus calling removes most of that background, so the variant stands out.
How LockSeq captures a target
From genomic DNA to a tagged, sequence-ready library
Every site in your panel goes through the same four steps, in one reaction.
01 · Hybridize
The probe finds its site
Both probe arms bind the sequence flanking the target, leaving a gap exactly over the site you want to read (Hardenbol et al. 2005).
02 · Gap-fill
The polymerase copies the site
A DNA polymerase fills the gap, copying the target sequence, including any edit, into the probe.
03 · Ligate
The ligase closes the circle
A DNA ligase seals the circle only when base pairing is exact. Each circle stands for one original DNA fragment and carries its own UMI.
04 · Amplify & sequence
A library from captured targets
Circles are amplified with sequencing handles and run on Illumina, MGI or Oxford Nanopore. Only captured targets make it into the library.
Validation
Benchmarked on NIST reference material
Five DNA mixtures from NIST's Genome in a Bottle, a 20-plex panel and 12 known variants, from single-base substitutions to indels of up to 54 bp. Measured frequencies, averaged over two replicates, matched expected values on both sequencing platforms, down to the lowest mixture tested.
0.175%
Lowest VAF tested
R² 0.99
Oxford Nanopore vs expected
R² 0.99
Illumina vs expected
12
known variants, 1 to 54 bp
What this means for your data
A clear answer for every site you nominate
Errors don't pass as edits
Consensus calling separates real variants from noise, so low-frequency events are reported with confidence. 0.1% VAF as standard, with each site’s limit set by its own coverage.
A 1–200 bp window, read in full
Each probe leaves a gap of up to 200 bp over the target and copies it in full, so substitutions and indels inside that window are read directly.
Every site clearly reported
Sites we can't design for are flagged before you send samples. Every other site gets a clear status in the report, including any that drop out in the lab.
Add sites as your program moves on
Probes are modular. Add or combine sites between development stages without redesigning the panel or losing performance.
Get in touch
Ready to run your off‑target confirmation panel?
Send us your nominated site coordinates and we'll scope the project within 24 hours.