NCM 2021: Phages, faeces, and PromethION: using nanopore to investigate the cattle slurry virome

Cows produce enormous amounts of waste, in the form of faeces, urine, and bedding, which is collected into a large slurry tank. The high phosphate and nitrate content of the waste means it can be used as an agricultural fertilizer. ‘Despite it being spread far and wide, we know incredibly little about its microbial composition’. Ryan took samples from a slurry tank and sequenced the viral fraction using short-read and long-read sequencing. Five viral DNA samples underwent short-read sequencing. These samples were also pooled, amplified, and sequenced using PromethION. Ryan compared the length of the viral genomes using short-read, long-read, and hybrid assemblies; the long-read sequences were approximately 2 kb longer than the short-read sequences. The average open reading frame predicted using PromethION technology was 85 amino acids, and this increased to 103 amino acids if polished with short-read sequences. Ryan found that polishing the short-read sequences removed erroneous stop codons, which helped with protein prediction and functional annotation. Comparison of the ‘read recruitment’ suggested that long-read sequencing gave ‘far better results’ than short-reads alone and could be further improved with polishing and de-replicating the hybrid contigs. When the read recruitment was normalised with phage abundance, it was possible to predict the diversity within the slurry tank. The long-read contigs estimated far higher biodiversity than short-read contigs, suggesting that just using short-read sequences could lead to underestimation of diversity within an environment. Ryan also estimated how complete the phage genomes were by comparing the short-read sequences and the polished PromethION results. A higher proportion of medium- and high-quality genomes were detected in the assembly containing the long reads (and a lower proportion of low-quality) compared with short-read data only. The slurry tank contained 7,682 viruses, and ‘98% of the tank was dominated by novel genera’. A number of interesting features which could alter the metabolism of the host were identified, including virulence factors and anti-microbial resistance genes which Ryan discussed further.