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

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. Availability and implementation: Puzzler is released into the public domain under 17 U.S.C. §105. Source code, documentation, and tutorials are available at https://github.com/merondun/puzzler and archived on Zenodo: https://doi.org/10.5281/zenodo.15733730 and https://doi.org/10.5281/zenodo.15693025. Pre-configured runtime environments including dependencies are provided via both a Conda environment (https://anaconda.org/heritabilities/puzzler) and an Apptainer hosted both on Zenodo and Sylabs (https://cloud.sylabs.io/library/merondun/default/puzzler).