HiFi reads combine long read lengths (>10 kb) and high accuracy (99.9%), which has enabled the complete and accurate characterization of whole genomes, including the first telomere-to-telomere assembly of a human genome. HiFi reads have simplified many bioinformatic analyses – efficient algorithms have been developed for read mapping, variant calling, and de novo genome assembly with HiFi reads. But obstacles have remained in preparing libraries, namely high DNA input requirements (5 µg per human genome) and a multi-step workflow requiring about 12 hours.
We have developed a HiFi library preparation workflow that reduces DNA input requirements, workflow steps, and end-to-end time. Steps have been combined, reagents provided as master mixes, and reaction times optimized. Size selection is performed with paramagnetic beads, which reduces workflow time and improves DNA recovery compared to size selection with gel cassettes. In total, the new workflow – based on PacBio SMRTbell Prep Kit 3.0 – reduces 10 steps to 7 and decreases workflow time from 12 hours to 6 hours.
We applied the workflow to generate libraries for a variety of samples including individual humans and multiplexed microbe strains. All samples produced high-quality libraries that resulted in high HiFi sequencing yields and excellent analysis results. The human sample HG002 achieved accurate variant calling (>99.9% SNV F1, >99.5% indel F1) and contiguous and accurate genome assembly (contig N50 >90 Mb, quality >Q50) at 32× coverage. All microbes assembled into complete genomes, with more than 375 Mb of total genome assembled from a single SMRT Cell.
This improved workflow supports high-throughput generation of libraries for PacBio HiFi sequencing including using liquid handling robots, which is required for rare disease and population genetics projects that are applying HiFi sequencing at new scales.