Rob James from the University of Warwick in the UK presented a session titled “From amplicons to metagenomes: long-read sequencing the environment” at London Calling 2019. James spoke about complex environmental matrices such as soil, sediment, and excreta that are often impure and biologically and chemically contaminated. Different extraction methods are used depending on the question being asked. The lab uses long-read sequencing to identify antimicrobial resistance and mobile genetic elements in complex environmental matrices. James described a project focusing on plasmid antimicrobial resistance associated with E. coli ST 131. They isolated the plasmid through conjugation and antibiotic selection. They then used Lambda exonuclease. The cleanup helped resolve the plasmid much better. The team fragmented with a Covaris gTube and assembled with Canu. They worked on two plasmids from isolates and were able to sequence the 91,523 bp and 101,530 bp plasmids. Plasmids have different clusters of genes associated with resistance. One plasmid does not seem to have conjugation-associated genes. The next project James described was on class I integrons from complex environmental communities from livestock in Pakistan. Building on this study, the team added sampling sites in the UK and Islamabad. They clustered sequences with vsearch and mapped with minimap2 to find a consensus with racon. This polishing procedure helped increase accuracy. Efflux pumps were abundant in different environments.
