We are going to use the Nanopore NO-MISS workflow. It will be used to sequence several microbial isolates. This is part of the Portable Genome Sequencing course. In preparation, this London Calling 2024 session seemed useful. Alex Trotter, Development Scientist, and Chris Alder, Bioinformatician, are both at Oxford Nanopore Technologies. They presented “How to sequence microbial isolates with the NO-MISS workflow.” This workflow is an end-to-end system for bacterial isolates. Trotter noted that this workflow can be adapted for 16S, viral amplicons, and even metagenomics. Long-read sequencing can help sequence through repetitive regions. Trotter emphasized that extraction methods have been optimized for ONT. After extraction, a cleanup may be performed. Bead-beating media may lead to smaller N50 values. ONT has reviewed and shared various protocols optimized for different organisms. There is even a protocol for fungal cells. Trotter recommended the RBK rapid barcoding kit. The preparation should take under an hour and can be for 24 of 96 barcodes. This kit works with a transposase to cleave and include adapters. Trotter noted that hours to one day of sequencing on a MinION flow cell should be sufficient. The MinION flow cells can be washed and reused. MinION and GridION devices can be used with the MinION flow cells. MinKNOW software is used for real-time base calling. EPI2ME is then used for bacterial genome assembly and annotation. Alder explained that Dorado base caller is available within MinKNOW. The EPI2ME desktop application is available for several operating systems. Workflows can be run locally or on the cloud. For the NO-MISS workflow, the wf-bacterial-genomes workflow is used. The minimum requirements are 16 Gb of RAM and 4 CPUs. Alder noted that the wf-bacterial-genomes workflow recommends at least 50X coverage. The inputs include FASTQ or BAM files, a reference if needed, and a sample sheet. The workflow can provide additional information with the isolates mode. Isolates mode includes multilocus sequence typing, antimicrobial resistance (AMR), and serotyping. Alder concluded by noting that additional information can be found on the webpage. We will use the NO-MISS workflow during the second lab session this semester.
