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. 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).
Meröndun et al. (Wed,) studied this question.