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ARS Home » Pacific West Area » Hilo, Hawaii » Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center » Tropical Plant Genetic Resources and Disease Research » Research » Publications at this Location » Publication #427500
Research Project: Conservation, Management, and Genetic Improvement of Tropical and Subtropical Fruit, Nut, and Beverage Crop Germplasm for the Pacific Region

Location: Tropical Plant Genetic Resources and Disease Research

Title: Puzzler: scalable one-command platinum-quality genome assembly from HiFi and Hi-C

Author
item MERONDUN, JUSTIN - Hawaii Agriculture Research Center
item Yu, Qingyi

Submitted to: Advances in Bioinformatics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/22/2025
Publication Date: 12/31/2025
Citation: Merondun, J., Yu, Q. 2025. Puzzler: scalable one-command platinum-quality genome assembly from HiFi and Hi-C. Advances in Bioinformatics. 6(1):vbaf329. https://doi.org/10.1093/bioadv/vbaf329.
DOI: https://doi.org/10.1093/bioadv/vbaf329

Interpretive Summary: High-quality, chromosome-level genome assemblies are critical for advancing research, from understanding evolutionary relationships to improving precision breeding programs. While new sequencing technologies have made it possible to generate highly accurate genome assemblies, the computational process remains complex, requiring specialized tools and expertise. There is a growing need for efficient, standardized, and user-friendly pipelines to streamline genome assembly. We developed a high-throughput, containerized pipeline, called Puzzler, for chromosome-scale de novo genome assembly. The pipeline offers optional modules for manual Hi-C refinement or entirely reference-free assembly. It includes built-in quality checks, such as Hi-C contact maps, BUSCO assessments, k-mer completeness analysis, and contamination screening. Puzzler has been successfully tested on a wide range of genome sizes, from very small (24 Mbp) to very large (6.4 Gbp), consistently producing highly complete and accurate results. To maximize accessibility, Puzzler is open-source and available on GitHub and Zenodo.

Technical Abstract: Motivation Chromosome-level assemblies are essential for modern genomics, from comparative genomics and evolutionary studies to precision breeding. While integrated HiFi and Hi-C data now enable accurate chromosome-scale genome assemblies, the bioinformatic process remains complex and involves specialized tools and expertise. With large-scale pan-genomic efforts requiring dozens to hundreds of platinum quality chromosome-scale genomes, there is a need for scalable, portable, and user-friendly pipelines that streamline and standardize high-quality genome assembly workflows. Results We introduce Puzzler, a containerized, scalable pipeline for chromosome-scale de novo genome assembly using PacBio HiFi and Hi-C data. Designed for portability and minimal user input, Puzzler automates contig assembly, duplicate purging, Hi-C-based scaffolding, and chromosome assignment via synteny, even with highly diverged reference taxa. Optional modules generate input files for manual Hi-C curation or operate reference-free. Quality control is integrated and includes Hi-C contact maps, BUSCO, yak k-mer completeness, and BlobTools contamination screening. A checkpointing system ensures that previously completed tasks are not re-executed, while a simple sample sheet input structure supports scalable batch processing. Puzzler has been validated on genomes ranging from 24 Mbp to 6.5 Gbp, delivering highly contiguous assemblies with <10'min of user input, enabling high-throughput platinum-quality genome assembly.