LockSeq Technical FAQ

This site provides detailed answers to the most frequent asked questions regarding the LockSeq platform.

🧪 Sample Requirements & Input

What DNA input amounts and concentrations are required?

For standard LockSeq processing, we typically require 100ng – 500ng of high-quality genomic DNA (gDNA) per sample. Ideally, samples should be provided at a concentration of >20ng/µL in 1x TE buffer or nuclease-free water.

Can you handle ultra-low input or fragmented DNA?

Yes. For rare or precious samples (e.g., primary cells or sorted populations), we have tested protocols for inputs as low as 5-10ng. For highly fragmented samples (e.g., FFPE or degraded material), please contact us and our technical team will get in touch with you to discuss this further,

Is LockSeq compatible with RNA targets?

Yes. While our current primary benchmarks focus on DNA/genotyping, the platform is based on padlock probe chemistry originally pioneered by Prof. Mats Nilsson for high-sensitivity RNA detection. We can quantify low-frequency transcripts or gene-fusion junctions from 100ng – 500ng of total RNA or ideally cDNA. If this is of interest to you, please contact us and the technical team will get in touch with you.

Do you support gRNA nomination / off-target site prediction?

Not currently as a primary service. LockSeq is a confirmation technology — it validates specific sites you've already nominated using upstream tools (GUIDE-seq, INDUCE-seq, CHANGE-seq, CIRCLE-seq, or in silico prediction).

If you provide us with your gRNA sequence, we can run in silico prediction algorithms (e.g., Cas-OFFinder and  CRISPOR) to help identify candidate sites if you don't have experimental nominations yet. Contact us to discuss your starting point.

🧬 Panel Design & Scalability

What coordinates do I need to provide for a custom panel?

We simply require the genomic coordinates of your intended edit and/or predicted off-target sites. Providing the gRNA sequence allows us to utilize in silico prediction algorithms if you require support in Nomination or don't have the on- and off-target coordinates.

What is the limit for multiplexing?

LockSeq is engineered to enable scalable gene-editing validation with minimal target dropout. Our current chemistry supports panels from 1- to 1,000-plex. If you require higher plexy, please get in touch with us and our technical team will get in touch with you.

Can I add new targets to an existing panel later?

Yes. One of our core advantages is Panel Flexibility. Because our ligation probes capture targets independently, you can "Mix and Match" coordinates. Unlike PCR assays that require complete redesigns to avoid new primer-dimers, you can simply add new probes to your existing LockSeq panel without sacrifising data integrity.

📈 Sensitivity & Performance

What is the verified Limit of Detection (LOD)?

Using NIST "Genome In A Bottle" (GIAB) standards, we have validated a Limit of Detection of 0.125% VAF (1:1,000).

How do you minimize the False Positive Rate (FPR)?

By integrating Unique Molecular Identifiers (UMIs) into the LockSeq probe design, our automated Python pipeline collapses reads and filters sequencing artifacts. This suppresses the noise floor to an extremely low FPR, ensuring that rare variants are biological truth backed by high-confidence reads, not stochastic noise.

What is the read coverage evenness (uniformity)?

LockSeq provides high uniformity across high-plex panels. Typically, >90% of targeted loci remain within a range of 0.5x to 2.5x of the mean depth. This high uniformity significantly reduces sequencing waste and ensures reliable variant calling across all sites in a single run.

How does LockSeq compare to multiplex PCR, such as rhAmpSeq from IDT?

The core difference is amplification strategy. rhAmpSeq uses multiplexed PCR, where dozens or hundreds of primer pairs compete in the same reaction. This creates amplification bias and target dropout based on published data.

LockSeq uses one probe per target, captured and circularized before amplification. Targets don't compete. This eliminates amplification bias and reduces dropout to 0–10% across all panels run to date.

Additionally, LockSeq's UMI-based consensus calling reduces the noise floor to resolve variants at 0.1% VAF with high confidence.

⏱️ Workflow & Automation

Is any specialized equipment required for the reagent kits?

Yes. The protocol is highly compatible with standard liquid handling systems (e.g., Opentrons, Hamilton, or Beckman Coulter).

What is the standard turnaround time (TAT)?

From receipt of DNA material in our Solna laboratory to the delivery of the final Variant Report, the standard TAT is 15 business days.

🖥️ Sequencing & Bioinformatics

Which sequencing platforms are compatible with LockSeq?

LockSeq is platform-agnostic. We produce inherently neutral circular templates that have been successfully benchmarked on Illumina and Oxford Nanopore Technologies chemistries. These are also the platforms we use for our end-to-end service.

However, LockSeq is in principle compatible with all available systems such as:

  • Short-read: Illumina and Element Biosciences.

  • Long-read: Oxford Nanopore.

  • Circular-based: PacBio and Complete Genomics.

What deliverables will I receive at the end of a study?

  • Comprehensive Variant Analysis Report: Detailed VAF distributions, indel profiling, and coverage uniformity charts.

  • Raw Data: Industry-standard FASTQ files for your own bioinformatic evaluation.

  • Consensus Alignment: UMI-corrected BAM files.