--- title: LockSeq Bioinformatics Pipeline | Countagen description: "How LockSeq turns sequencing reads into a per-site call: UMI consensus, raw-read confirmation, control comparison and one interactive report." --- [![Logo\_horizontal\_colored logo with black text](https://www.countagen.com/hubfs/Logo_horizontal_colored%20logo%20with%20black%20text.png)](https://www.countagen.com/?hsLang=en) - [Technology](https://www.countagen.com/technology?hsLang=en) - [Bioinformatics](https://www.countagen.com/bioinformatics) - [Sample Report](https://www.countagen.com/lockseq-sample-report?hsLang=en) - [Resources](https://www.countagen.com/resources?hsLang=en) - [FAQ](https://www.countagen.com/faq?hsLang=en) - [About Us](https://www.countagen.com/about-us?hsLang=en) [Request a quote](https://www.countagen.com/contact?hsLang=en) Bioinformatics # From FASTQ to confident variant calls The LockSeq pipeline checks each variant twice, in raw reads and in UMI consensus reads, then against your untreated control. You get one interactive report with a status for every site in your panel. [Explore the sample report](https://www.countagen.com/lockseq-sample-report?hsLang=en)[Request a quote](https://www.countagen.com/contact?hsLang=en) RAW READS AT ONE SITERaw layerevery read, as sequencedConsensus layerone read per UMI familyCALLConfirmed, treated-associatedIn both layers · absent from the untreated controlTrue variantRandom error 0.1% VAF Standard limit of detection; each site’s limit is set by its own coverage 3 reads Minimum UMI family size for a consensus read 30k / 3k Standard sequencing depth per site (raw / consensus) 3 platforms Illumina, MGI and Oxford Nanopore, same pipeline The pipeline ## Six modules, from FASTQ to report Every run goes through the same steps, from a 20-site panel to one designed for 1,000+ sites, so results compare across panels, sequencers and time. 01 · Read QC ### Clean reads in, run quality on record Trims adapters, filters low-quality reads and writes a quality summary for every sample in the run. 02 · UMI extraction ### Each read keeps its molecule tag Reads the unique molecular identifier off every read and stores it with the read before anything is aligned. 03 · Mapping ### Reads placed on their target site Aligns reads to the reference sequence of each target site, taken from your organism's reference genome (for example GRCh38 for human), then sorts and indexes the alignments. 04 · Consensus building ### One consensus read per original molecule Groups reads that share a UMI. A family of at least 3 reads becomes one consensus read, so errors that appear in a single read drop out. 05 · Variant annotation Runs on raw and consensus reads ### SNVs and indels, allele by allele Aligns every read at the site against its reference and annotates substitutions, insertions and deletions per allele and per sample. 06 · Comparison & report ### Treated vs control, both layers, one report Checks each variant against both evidence layers and your untreated controls, assigns every site a data status and writes the interactive report. Two layers of evidence ## A variant is confirmed only when both layers agree Raw reads show everything the sequencer saw. Consensus reads keep only what each original molecule carried. A true variant is in every read from its molecule, so it survives consensus building. A random PCR or sequencing error is in only some of those reads, so the consensus drops it. The pipeline reports the variant frequency from the consensus layer and needs matching evidence in the raw reads before it calls a variant confirmed. Variants below the raw threshold, or found only in raw reads, stay visible with a flag but aren't counted as confirmed. Consensus-only variants are removed as likely artifacts and listed in an audit file. A site needs at least 1,000 raw and 30 consensus reads to be called. Sites below that are flagged in the report, not left out. | Evidence flag | Consensus | Raw | | --- | --- | --- | | ConfirmedVariant in both layers Reported variant | ✓ | ✓ | | Raw support below limitRaw evidence matches but sits below the raw reporting threshold Flagged, not counted | ✓ | ~ | | No raw supportConsensus variant not found in the raw reads Removed as artifact | ✓ | – | | Not confirmedRaw-read variant with no consensus evidence Flagged, not counted | – | ✓ | What consensus does to the data Raw readsThe variant hides in NGS background 0.0%0.1%0.2%0.3%variantPosition across the site Consensus readsThe true variant stands out clearly 0.0%0.1%0.2%0.3%variantPosition across the site 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. Checked against your control ## Variants in your control are kept apart from edits Every genome differs from the reference at millions of positions. So each treated sample is compared with your own untreated control as well as the reference genome. A variant at a similar frequency in treated and control samples is called in control. Only treated-associated variants count as editing signal. LockSeq report · Variant Frequency Overview ![Variant frequency heatmap: three on-target sites at about 69 to 78 percent and one off-target site at about 0.2 percent in the three treated samples, 0 percent in controls; two off-target sites show similar frequencies around 0.5 percent in treated and control samples and are called in control](https://www.countagen.com/hubfs/report-heatmap-2026-10.png) Sample reportThe three on-target sites and one off-target site, DP-187, carry the edit in treated samples only. DP-012 and DP-069 show the same low-level variant in treated and control samples, so they're called in control and not counted as edits. Synthetic demo data. Allele-level view ## See the alleles behind every number For each site and sample, the report shows the allele mix behind the frequency: which deletions, insertions and substitutions, how often, and from how many consensus reads. Any call can be checked by eye. Both layers have their own allele plots, so you can put the raw and consensus view of the same site side by side. LockSeq report · Consensus allele plot ![Consensus allele plot for one on-target site: reference sequence on top, 29 percent unedited reads, then deletions of 2 to 6 base pairs at the cut site at decreasing frequencies, and one G to T substitution](https://www.countagen.com/hubfs/report-allele-plot-2026-10.png) Sample reportOne on-target site in one treated sample: 29% of consensus reads unedited, the rest a mix of deletions at the cut site. Synthetic demo data. Reporting rules ## Every site in your panel gets a status The thresholds are the same on every run and printed in every report, so you know exactly why a site was called the way it was. 30consensus reads per site for an interpretable call 1,000raw reads per site for usable raw evidence 3 / Nlower reportable limit, where N is the consensus depth. 3,000 consensus reads gets you to 0.1% VAF. ~80%of sites typically within 80% of the mean read depth, so you don't over-sequence the panel to reach the weakest site Reportable Enough consensus support to report a variant frequency. Raw evidence only Raw reads show evidence, but consensus support is below the reporting guide. Low support Consensus depth below the interpretable threshold. Dropout No usable consensus reads for one or more samples. Probe review A capture or alignment pattern, such as reads from a second locus, flagged for manual review. What you get back ## The report and your raw data ### Interactive HTML report One self-contained file that opens in any browser. Filter by coverage, compare against controls, open the reads and export tables as CSV. ### FASTQ and BAM files Your raw and aligned data, ready for your own team to reanalyze in-house. ### Analysis included No separate software, analysis or per-run fees. The pipeline is part of the project price. ### 15 business days From the day your DNA arrives in our lab in Solna, Stockholm to the delivered report. See it for yourself ## Open a full LockSeq report Click through the sample report, or send us your nominated sites and we'll scope your panel within 24 hours. [Explore the sample report](https://www.countagen.com/lockseq-sample-report?hsLang=en)[Request a quote](https://www.countagen.com/contact?hsLang=en) [![Countagen\_Logo\_White](https://www.countagen.com/hubfs/Logo_horizontal_colored%20logo%20with%20white%20text.png)](https://www.countagen.com?hsLang=en) | Countagen AB Nobels väg 16 171 65 Solna Sweden | Registered supplier on [Scientist.com](https://app.scientist.com/providers/countagen-ab) [![Scientist.com](https://www.countagen.com/hs-fs/hubfs/Scientist-com.png?width=1244&height=259&name=Scientist-com.png)](https://app.scientist.com/providers/countagen-ab) | | --- | --- | ### LockSeq - [Overview](https://www.countagen.com?hsLang=en) - [Technology](https://www.countagen.com/technology?hsLang=en) - [Bioinformatics](https://www.countagen.com/bioinformatics) - [Sample report](https://www.countagen.com/lockseq-sample-report?hsLang=en) ### Resources - [Resources](https://www.countagen.com/resources?hsLang=en) - [FAQ](https://www.countagen.com/faq?hsLang=en) ### Company - [About us](https://www.countagen.com/about-us?hsLang=en) - [Contact](https://www.countagen.com/contact?hsLang=en) © 2026 Countagen AB All rights reserved [Terms of Use](https://www.countagen.com/terms-of-use?hsLang=en) [Privacy Policy](https://www.countagen.com/privacy-policy?hsLang=en) [Cookie Policy](https://www.countagen.com/cookie-policy?hsLang=en) [Service Terms & DPA](https://www.countagen.com/legal?hsLang=en) [Powered by Atlas - a B2B SaaS HubSpot theme](https://www.kalungi.com/atlas-hubspot-theme-for-b2b-saas-software)