As part of the Knowledge Exchange series of lessons, John Beaulaurier from Oxford Nanopore Technologies spoke about assembly. The session’s title is “No assembly required: nanopore sequencing complete virus genomes from microbial communities.” The session was recorded on September 30, 2019. Beaulaurier worked with academic collaborators on this project. Beaulaurier explained that marine microbial populations are often shaped by bacteriophage populations. Phages help release 10 billion tons of carbon each day through bacterial lysis and are a major source of gene diversity through horizontal gene transfer in oceans. Previously, fosmids were used to clone up to 40 kb genomes. Short read sequencing approaches often result in fragmented/incomplete genomes. Three samples came from a station Aloha. Ed Delong from the University of Hawaii and team helped sample. Station allowed for sampling three different depths. DNA was extracted using the QIAGEN genomic tip kit. Libraries were prepared with the LSK 109 kit and loaded onto three flow cells run on a GridION. The sequencing output resulted in “redundant observations of the entire sequence.” Reads with direct terminal repeats were identified by “align first & last 20% of each read (minimap2).” Counts of terminal repeats were collected. A table was generated and then dimensionality reduction UMAP was used. The system was trained on a dataset from metagenomic fosmid sequencing. These phage genomes include 193 marine phage genomes. Reads were simulated with about 50 reads per viral genome. As depth increases, more viral diversity was obtained. Full length genomes identified cyclically permuted genomes and differences that would not have been revealed with short-read sequencing. Phage-inducible chromosomal islands (PICI) are mobile DNA elements that hijack phage packaging machinery. This approach could be applied to other viruses and sample types. Another application could be to discover plasmids in metagenomic samples.
