Haplotype-resolved assembly of a pig genome using single-sperm sequencing

Abstract

Single gamete cell sequencing together with long-read sequencing can reliably produce chromosome-level phased genomes. In this study, we employed PacBio HiFi and Hi-C sequencing on a male Landrace pig, coupled with single-sperm sequencing of its 102 sperm cells. A haplotype assembly method was developed based on long-read sequencing and sperm-phased markers. The chromosome-level phased assembly showed higher phasing accuracy than methods that rely only on HiFi reads. The use of single-sperm sequencing data enabled the construction of a genetic map, successfully mapping the sperm motility trait to a specific region on chromosome 1 (105.40-110.70 Mb). Furthermore, with the assistance of Y chromosome-bearing sperm data, 26.16 Mb Y chromosome sequences were assembled. We report a reliable approach for assembling chromosome-level phased genomes and reveal the potential of sperm population in basic biology research and sperm phenotype research.

Fig. 1. Schematic diagram of sperm haplotype inference.

Numbers 1-8 represent the chromosomes of No.1-8 sperms, and the parental genotype was indicated by A, B, and orange and blue colors, respectively. The black horizontal lines represent false recombination sites in the population. a The real chromosome recombination map of the eight sperm. b The recombination map after genotyping using sperm No.1 as the reference. The same genotype with No.1 sperm was marked as orange, and the different genotype was marked as blue. False recombination occurs at the horizontal line with all sperm (except No. 1) recombined at this position, which was caused by the real recombination of No. 1 sperm. c The recombination map of No. 2-8 sperm after the false recombination site correction. d The recombination map after the correction of all sperm. e The complete haplotype path is based on the population genotype information, which was indicated in dark orange and dark blue.

Fig. 2. Inferring parental haplotype using sperm population.

a The preliminary recombination map inferred by using sperm S18-162 as the reference, the chromosomes are divided by the white vertical lines, and the vertical black dotted lines represent the potential false recombination site. b The final recombination map after correcting the false recombination sites and filtering suspicious recombination.

Fig. 3. Comparison of different strategies of phased assembly.

a–c The phased marker distribution in Dipasm assembly (a), Falcon phase assembly (b), and single sperm-based assembly (c). d Collinearity analysis of the single sperm assembly and the Duroc genome.

Fig. 4. Comparison of genetic map and physical map.

The X axes represents the physical location, and the Y axes represents the genetic distance.

Fig. 5. QTL mapping of sperm activity traits.

The X axes represent the chromosome, and the Y axes represent the LOD values. The dashed line represents the significant threshold of LOD at 2.5. LOD is the log10 likelihood ratio comparing the hypothesis of a QTL at position versus that of no QTL.