Skim-Nanopore sequencing for routine genomic evaluation and bacterial pathogen detection in cattle

Abstract

Context Genotyping-by-sequencing, the use of sequence reads to genotype single-nucleotide polymorphisms (SNPs), has seen an increase in popularity as a tool for genomic prediction. Oxford Nanopore Technologies (Nanopore) sequencing is an emerging technology that produces long sequence reads in real-time. Recent studies have established the ability for low-coverage Nanopore sequence data to be used for genomic prediction. However, the value proposition of Nanopore sequencing for individuals could be improved if both genotyping and disease diagnosis are achieved from a single sample. Aims This study aimed to demonstrate that Nanopore sequencing can be used for both rapid genotyping and as a disease diagnostic tool using the same sample in livestock. Methods Total DNA extracts from nasal swabs collected from 48 feedlot cattle presenting with clinical signs of bovine respiratory disease (BRD) were sequenced using the Nanopore PromethION sequencer. After 24 h of sequencing, genotypes were imputed and genomic estimated breeding values (GEBVs) for four traits were derived using 641 163 SNPs and corresponding SNP effects. These GEBVs were compared with GEBVs derived from SNP array genotypes and calculated using the same SNP effects. Unmapped sequence reads were classified into taxa using Kraken2 and compared with quantitative real-time polymerase chain reaction (qPCR) results for five BRD-associated pathogens of interest. Key results Sequence-derived genotypes for 46 of the 48 animals were produced in 24 h and GEBV correlations ranged between 0.92 and 0.94 for the four traits. Eleven different BRD-associated pathogens (two viruses and nine bacterial species) were detected in the samples using Nanopore sequence data. A significant (P < 0.001) relationship between Nanopore and qPCR results was observed for five overlapping species when a maximum threshold cycle was used. Conclusions The results of this study indicated that 46 cattle genomes can be multiplexed and accurately genotyped for downstream genomic prediction by using a single PromethION flow cell (ver. R9.4) in 24 h. This equates to a cost of AUD35.82 per sample for consumables. The concordance between qPCR results and pathogen proportion estimates also indicated that some pathogenic species, in particular bacterial species, can be accurately identified from the same test. Implications Using Nanopore sequencing, routine genotyping and disease detection in livestock could be combined into one cost-competitive test with a rapid turnaround time.