Background

KaryoSolver is our proprietary solution for molecular karyotyping from low-pass whole genome sequencing (lpWGS), tested on Nanopore data. It leverages very fast/real-time sequencing to generate genome-wide copy number profiles, identifying large chromosomal abnormalities, microdeletions/duplications, and mosaicism.

Unlike traditional array-CGH, KaryoSolver provides:
– Scalable resolution in short time (hours, depending on sequencing time).
– Mosaic fraction estimation, enabling detection of clonal heterogeneity.
– Genome-wide coverage without probe design limitations, minimizing blind spots.

KaryoSolver module is part of 4eVAR platform, and builds on the academic framework of Nano-GLADIATOR (Magi et al., 2019), which demonstrated that shallow Nanopore sequencing can deliver array-like CNV profiles with increasing resolution as sequencing progresses.

How It Works

  • Sequencing input: Nanopore FASTQ files at 0.5–4× coverage.
  • Connect to 4evar.4bases.ch and create a new account
  • Download the Client (compatible with all platforms) and install it ( Minimum requirements is 4GB RAM) 
  • Download the reference folder
  • Alignment: long-read mapper (e.g., minimap2 ONT preset) against the GRCh38 no-alt reference (given with the tool).

Figure 1: 4eVAR – KaryoSolver dashboard

  • Open Karyosolver dashboard and Load data in single mode or Batch mode

Figure 2: KaryoSolver modal to load BAM file.

  • Wait the BAM verification and processing, and then you will find your new Analysis in Load Experiment. Select it and press RUN.

Figure 3: “Waiting for Data Elaboration” window.

In about a minute you will have genome-wide binning and segmentation with three main parameters:
   • window_size: bin size (smaller = higher resolution, more noise).

   • Omega (OM): segmentation sensitivity (higher = more sensitive, noisier).

   • Filtered Windows (FW): minimum consecutive bins for a CNV call (higher = fewer noisy calls).

Figure 4: KaryoSolver dashboard.

Output: an interactive molecular karyotype profile, with tables and plots for exploration and annotation.

Sample Preparation

For best results, we recommend following the 4bases Technote for KaryoSolver sample preparation (RUO version):

  • High-quality, high molecular weight genomic DNA.
  • Standard ONT Rapid Sequencing Kit V14 – gDNA (SQK-RAD114) or Rapid sequencing DNA V14 – barcoding ( SQK-RBK114.24 or SQK-RBK114.96) or optimized multiplex-ready 4bases solution comprising of WGS preparation kit (RDC3290-16 or RDC3290-96), Nanoadapter (M1050-6 or M1050-24) in combinantion with ONT Ligation sequencing DNA V14 (SQK-LSK114)
  • Optimal input and clean-up steps as described in the official Technote.
  • Avoid degraded samples and ensure QC prior to sequencing.

This ensures consistent coverage profiles and reliable CNV detection.

Sequencing Time vs. Resolution

KaryoSolver provides progressive resolution as sequencing time increases. This makes it directly comparable to Agilent array-CGH platforms, but with faster turnaround and flexible resolution.

Platform

Typical Resolution

Processing/Sequencing Time

Turnaround

60K CGHarray

~150 kb

24–40 h hybridization + 6–8 h prep

2–3 days

400K CGHarray

~30–50 kb

24–40 h hybridization + 6–8 h prep

2–3 days

1M CGHarray

~20–30 kb

24–40 h hybridization + 6–8 h prep

2–3 days

KaryoSolver

10Mb >2h

100kb>6h

30kb>24h

Fast sequencing

Same day (6–24 h)*

* Standard ONT rapid or optimized multiplex-ready 4bases RDC3290 preparation kit with ligation library prep V14.

Interactive & Refinement Features

KaryoSolver allows interactive exploration of CNVs:
– A whole-genome plot displays normalized log2 ratios across all chromosomes for rapid visualization of alterations.
– Clicking on a chromosome segment in the plot highlights the CNV in detail with annotation.
– The Cellular Fraction Threshold can be adjusted to hide low-fraction events and focus on clinically relevant CNVs.
– Users can refine results by tuning OM and FW values iteratively to balance sensitivity against noise.

Interpretation & Clinical Guidelines

KaryoSolver is fully integrated with ClinGen resources for ACMG-compliant CNV interpretation. CNVs identified are annotated with overlapping known pathogenic regions, dosage-sensitive genes, and databases such as DECIPHER and ClinVar. This ensures that each finding is contextualized within clinically validated knowledge.

Technical Notes

Normalization and log2 ratio calculation are at the core of KaryoSolver’s CNV calling:
– Each genomic window (bin) is normalized against expected read counts.
– Log2 ratios are computed as log2(observed/expected coverage).
– Typical thresholds:
   • ~0.0 → diploid (2 copies).
   • –0.5 → heterozygous deletion (1 copy).
   • –1.0 → homozygous deletion (0 copies).
   • +0.58 → single duplication (3 copies).

Mosaicism: refers to the presence of two or more genetically distinct cell populations derived from a single zygote within the same individual. In KaryoSolver, mosaicism is inferred when the log2 ratio deviation is intermediate (e.g., –0.25 for a 50% mosaic heterozygous deletion).

Advantages of KaryoSolver

  • Real-time results: large CNVs visible within 1–2 hours of sequencing.
  • Adjustable resolution: allow the run to continue for higher sensitivity.
  • Mosaicism detection: estimate clonal fractions directly from log2 ratios.
  • Interactive visualization: genome-wide plots with clickable chromosome segments.
  • ClinGen/ACMG integration: annotations aligned with clinical guidelines.
  • User-friendly interface for both single-sample and batch analysis.
  • Integrated with 4eVAR: streamlined access and project management.

Application Notes

Reference (required)

Use GRCh38 no-alt analysis set (UCSC IDs) to align with ACMG guidance for large CNVs.

Source: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_for_alignment_pipelines.ucsc_ids/

Files: the FASTA (…no_alt_analysis_set.fasta or .fna) and its index (.fasta.fai or .fna.fai).

You may also use the bundled copies shipped with the local client in /data folder:

/data/references/hg38/GCA_000001405.15_GRCh38_no_alt_analysis_set.fasta

/data/references/hg38/GCA_000001405.15_GRCh38_no_alt_analysis_set.fasta.fai

Alignment (concepts)

  • Align ONT FASTQ with a long-read mapper (e.g., minimap2 ONT preset) against the exact reference above.

  • The resulting BAM must be coordinate-sorted and indexed (.bai) using (e.g., samtools view, pysam…).

  • Keep .bam and .bai in the same folder.

  • Ensure header/reference consistency (the BAM must match the GRCh38 no-alt build used).

Why this matters

Correct reference choice and sorted/indexed BAMs are essential for robust read-counting, segmentation, and ACMG-consistent large-CNV interpretation.

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TECHNICAL NOTE FOR KARYOSOLVER PRO