Long-read Pore-C shows the 3D structure of the cattle genome

Abstract

Context Recent advances in molecular technology have allowed us to examine the cattle genome with an accuracy never before possible. Genetic variations, both small and large, as well as the transcriptional landscape of the bovine genome, have both been explored in many studies. However, the topological configuration of the genome has not been extensively investigated, largely due to the cost of the assays required. Such assays can both identify topologically associated domains and be used for genome scaffolding. Aims This study aimed to implement a chromatin conformation capture together with long-read nanopore sequencing (Pore-C) pipeline for scaffolding a draft assembly and identifying topologically associating domains (TADs) of a Bos indicus Brahman cow. Methods Genomic DNA from a liver sample was first cross-linked to proteins, preserving the spatial proximity of loci. Restriction digestion and proximity ligation were then used to join cross-linked fragments, followed by nucleic isolation. The Pore-C DNA extracts were then prepped and sequenced on a PromethION device. Two genome assemblies were used to analyse the data, namely, one generated from sequencing of the same Brahman cow, and the other is the ARS-UCD1.2 Bos taurus assembly. The Pore-C snakemake pipeline was used to map, assign bins and scaffold the draft and current annotated bovine assemblies. The contact matrices were then used to identify TADs. Key results The study scaffolded a chromosome-level Bos indicus assembly representing 30 chromosomes. The scaffolded assembly showed a total of 215 contigs (2.6 Gbp) with N50 of 44.8 Mb. The maximum contig length was 156.8 Mb. The GC content of the scaffold assembly is 41 ± 0.02%. Over 50% of mapped chimeric reads identified for both assemblies had three or more contacts. This is the first experimental study to identify TADs in bovine species. In total, 3036 and 3094 TADs across 30 chromosomes were identified for input Brahman and ARS-UCD1.2 assemblies respectively. Conclusions The Pore-C pipeline presented herein will be a valuable approach to scaffold draft assemblies for agricultural species and understand the chromatin structure at different scales. Implications The Pore-C approach will open a new era of 3D genome-organisation studies across agriculture species.