Long-Read Low-Pass Sequencing for High-Resolution Trait Mapping

Accelerating crop improvement is critical to meeting food security demands in a changing climate. Long-read sequencing offers advantages over short-reads in resolving structural variations (SVs) and aligning to complex genomes, but its high cost has limited adoption in breeding programs. Here, we develop a high-throughput, scalable approach for long-read low-pass (LRLP) sequencing and variant analysis with PacBio HiFi reads, and apply it to trait mapping in a complex tetraploid peanut (Arachis hypogaea) genome multi-parent advanced generation intercross. We analyze LRLP using both a single reference genome and a pangraph, using both proprietary and open-source tools to analyze SVs and coverage. An increased number of variants are consistently called for LRLP data compared to short-read data. At 1.63x average depth, LRLP sequencing covered 55% of the genome and 58% of gene space, outperforming 1.68x depth short-read low-pass sequencing, which achieved only 17% and 11%, respectively. Enhanced data retention after filtering for probabilistic misalignment and an ~8.5x decrease in cost per value further demonstrated LRLPs efficacy. Our results highlight LRLP sequencing as a scalable, cost-effective tool for high-resolution trait mapping, with transformative potential for plant breeding and broader genomic applications.