Natalie Ring from The Roslin Institute in the UK presented at London Calling 2022 on “DoGsTAils: developing nanopore-based diagnostic approaches for the effective antimicrobial stewardship of canine infections.” Ring spoke about skin and urinary tract infections in dogs and how they can be antibiotic resistant. It also often takes two days to obtain results. Thus, Ring and colleagues wanted a quick workflow to collect clinical samples and obtain a candidate organism. The group started by reviewing skin infection and UTI data from the University of Edinburgh Small Animal Hospital from 2015-2019. The organisms are almost evenly split between Gram positive and Gram negative organisms. The team used the QIAGEN MagAttract HMW kit + Metapolyzyme lysis kit with a mixture of six enzymes. Post-extraction, the team used ProNEX beads. The extraction process takes about 2-3 hours. They started using the Rapid PCR Barcoding kit and MIN-106D flow cells but it amplified dog DNA too! Thus, they used the Rapid Barcoding kit instead. They started by using the MinION M1kc. The limits of detection were tested with dilutions of E. coli to determine how much DNA they needed to load to use the kit. They found that 0.07 ng/ul was enough to identify the organism… however they then learned that this organism may not be the best choice as it is often a contaminant. The team has tested twenty samples and been able to detect 19/20. Ring did a flow cell washing experiment and sequenced until reaching 200 Mb. They used the Rapid Barcoding kit and different barcodes. Ring was able to use the flow cell for eight wash/run cycles with 99% correct barcode. Ring has done a run on a dog sample. They noted that a twenty minute spin helps increase DNA yield from samples. The team is now using a GridION and hopes to automate the process. I am excited they mentioned that on the GridION they can do real-time super accurate base calling and analysis. I also didn’t know about the Promega ProNEX beads. I wonder how compatible they are with library prep kits. Ring and colleagues did a careful and systematic confirmation of their workflow.
