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The combination of ultra-long (UL) Oxford Nanopore Technologies (ONT) sequencing reads with long, accurate Pacific Bioscience (PacBio) High Fidelity (HiFi) reads has enabled the completion of a human genome and spurred similar efforts to complete the genomes of many other species. However, this approach for complete, “telomere-to-telomere” genome assembly relies on multiple sequencing platforms, limiting its accessibility. ONT “Duplex” sequencing reads, where both strands of the DNA are read to improve quality, promise high per-base accuracy. To evaluate this new data type, we generated ONT Duplex data for three widely studied genomes: human HG002, Solanum lycopersicum Heinz 1706 (tomato), and Zea mays B73 (maize). For the diploid, heterozygous HG002 genome, we also used “Pore-C” chromatin contact mapping to completely phase the haplotypes. We found the accuracy of Duplex data to be similar to HiFi sequencing, but with read lengths tens of kilobases longer, and the Pore-C data to be compatible with existing diploid assembly algorithms. This combination of read length and accuracy enables the construction of a high-quality initial assembly, which can then be further resolved using the UL reads, and finally phased into chromosome-scale haplotypes with Pore-C. The resulting assemblies have a base accuracy exceeding 99.999% (Q50) and near-perfect continuity, with most chromosomes assembled as single contigs. We conclude that ONT sequencing is a viable alternative to HiFi sequencing for de novo genome assembly, and provides a multirun single-instrument solution for the reconstruction of complete genomes.
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The revised reference genome of the leopard gecko ( Eublepharis macularius ) provides insight into the considerations of genome phasing and assembly
Abstract Genomic resources across squamate reptiles (lizards and snakes) have lagged behind other vertebrate systems and high-quality reference genomes remain scarce. Of the 23 chromosome-scale reference genomes across the order, only 12 of the ~60 squamate families are represented. Within geckos (infraorder Gekkota), a species-rich clade of lizards, chromosome-level genomes are exceptionally sparse representing only two of the seven extant families. Using the latest advances in genome sequencing and assembly methods, we generated one of the highest-quality squamate genomes to date for the leopard gecko, Eublepharis macularius (Eublepharidae). We compared this assembly to the previous, short-read only, E. macularius reference genome published in 2016 and examined potential factors within the assembly influencing contiguity of genome assemblies using PacBio HiFi data. Briefly, the read N50 of the PacBio HiFi reads generated for this study was equal to the contig N50 of the previous E. macularius reference genome at 20.4 kilobases. The HiFi reads were assembled into a total of 132 contigs, which was further scaffolded using HiC data into 75 total sequences representing all 19 chromosomes. We identified 9 of the 19 chromosomal scaffolds were assembled as a near-single contig, whereas the other 10 chromosomes were each scaffolded together from multiple contigs. We qualitatively identified that the percent repeat content within a chromosome broadly affects its assembly contiguity prior to scaffolding. This genome assembly signifies a new age for squamate genomics where high-quality reference genomes rivaling some of the best vertebrate genome assemblies can be generated for a fraction of previous cost estimates. This new E. macularius reference assembly is available on NCBI at JAOPLA010000000.
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- Award ID(s):
- 1657662
- PAR ID:
- 10405812
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Journal of Heredity
- ISSN:
- 0022-1503
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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