TaKoHigh: Accurate variant calling and phasing from PCR-based long-read sequencing data

Overview
TaKoHigh is an amplicon-aware software tool that enables accurate variant calling and phasing on real-world PCR-based long-read sequencing data. It is robust to common PCR-derived artifacts and does not require special experimental protocols, making it compatible with standard laboratory workflows. This tool advances reliable interpretation of long-read amplicon data in both clinical and research settings.

Citation
Eura Y, Takahashi A, Ye S, Sakai K, Matsumoto M, Kokame K. TaKoHigh enables accurate variant calling and phasing from PCR-based long-read sequencing. Jxiv, 2025

Quick Start
1. Prepare input files
BAM File (.bam): Produce with Minimap2 or a similar aligner. An index file (.bai) is not necessary. One BAM file containing reads from one or more amplicons is sufficient.

Amplicon Definition File (tab-delimited .txt): Four columns in this order:
amplicon_id | chrom | start | end
Chromosome names must match the BAM/reference.

(Example)
PCR1	chr9	133423438	133433779
PCR2	chr9	133429050	133438489
PCR3	chr9	133434246	133443653

Reference Sequence File (.rse and .cti): Required for the first run. In the app, open Other -> Create Reference File to download the specified reference (e.g., GRCh38) from NCBI, then Other -> Create DB to convert; this produces two files (.rse and .cti).

2. (optional) Excluded Variants File
To mask suspected sequencing error sites, provide a tab-delimited text file (.txt) with columns:
chrom | pos

(Example)
chr9	133424549
chr9	133420161
chr9	133435357

3. Run TaKoHigh
Select BAM File (.bam), Amplicon Definition File (tab-delimited .txt), Reference Sequence File (.rse and .cti), Output File, and Excluded Variants File (optional), then click Analysis.

Output
Results are written to a CSV file with two sections:

Phased haplotypes (top): a list of phased haplotypes in genomic order (phase sets on contiguous segments). If two or more segments appear (i.e., blank lines separate segments), cross-amplicon phasing is not fully connected. Designing additional amplicons that bridge the gap and re-running TaKoHigh may connect the segments.

Per-amplicon variant table (bottom): variant calls and annotations for each amplicon.

Key Annotations
StrandBias — Heterozygous SNVs with a significant strand imbalance between reference and alternate reads (p < 1e-4)

StrandBias|NotVariant — Reference homozygous sites exhibiting strand bias

UnusedForPhasing — Sites not used for phasing (e.g., second allele cannot be determined).

Supported Operating Systems
macOS: Apple silicon (M1 or later). Intel Macs are not supported.
Windows: Windows 10/11 (64-bit).
We recommend keeping your OS up to date for security and bug fixes.

Notes and Tips
Use the same reference build (e.g., GRCh38) and chromosome naming across the BAM, amplicon file, and reference bundle.
If phasing remains incomplete, add one or more overlapping amplicons that include a heterozygous SNV near the gap and re-run TaKoHigh.
For reproducibility, keep the .bam, .txt, .cti, and .rse files alongside the output .csv file.

License and Permissions
Copyright © 2025 National Cerebral and Cardiovascular Center (NCVC), Japan. All rights reserved.

Academic (non-commercial) use:
TaKoHigh may be used for academic, non-commercial research and educational purposes free of charge, provided that this copyright notice and the citation to the TaKoHigh paper are retained in all copies and substantial portions of the software and associated documentation.

Commercial use:
Any commercial use (including use in paid services, products, or internal workflows at for-profit entities) requires prior written permission from the authors/NCVC. Please contact the corresponding author (see the manuscript) to request a license.

Redistribution:
Redistribution is permitted for academic, non-commercial purposes only, provided this License and Permissions section remains intact. Commercial redistribution (e.g., bundling with paid software or services) requires prior written permission.

Attribution:
Publications and derivatives using TaKoHigh must cite the TaKoHigh paper.

No endorsement:
The names "TaKoHigh" and "NCVC" may not be used to endorse or promote products derived from this software without specific prior written permission.

Disclaimer:
TaKoHigh is provided "as is", without warranty of any kind (including merchantability, fitness for a particular purpose, and non-infringement). The authors and NCVC assume no liability for any damages arising from use of this software. Research use only; not a medical device.