## Draft Genome Sequencing of Bacteria and Fungi Cultured by Undergraduate Students in Fairbanks, Alaska * Eric Collins, College of Fisheries and Ocean Sciences, University of Alaska Fairbanks, Fairbanks, Alaska, USA * Mary Beth Leigh, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, USA Abstract Undergraduate students each cultured, sequenced, and analyzed the complete genome of a microorganism as part of the General Microbiology course at the University of Alaska Fairbanks. Common skin microbiota were isolated most frequently, but some rare taxa, including several Deinococcus, were isolated as well. Microbial Resource Announcement In order to introduce undergraduate students to modern day genomics techniques, each student in the 300-level General Microbiology course at the University of Alaska Fairbanks was assigned to isolate a microorganism from the environment, phenotypically characterize it, extract DNA from it, submit it for complete genome sequencing, and analyze the resulting draft genome sequence using web-based bioinformatics tools. Students enrolled in the course in 2017—2019 were directed to culture microorganisms from any environmental source but were discouraged from using sources likely to host pathogens. (Complete lab section protocols and bioinformatics analysis scripts for the methods are available at http://github.com/rec3141/biol342). Environmental material was plated onto TSA plates (in 2017) or either TSA or R2A plates (in 2018—2019) and colonies were streaked to presumptive isolation (a minimum of 3 streaks) at either room temperature or 37°C. In practice, 69% of cultures were pure, 20% were co-cultures containing 2 organisms, and 11% were co-cultures of 3 or more organisms. Cultures were subjected to numerous phenotypic tests including Gram-staining, antibiotic susceptibility, fluid thioglycollate test, oxidase test, catalase test, and API 20E test strips. DNA was extracted using the MoBio PowerSoil DNA Isolation Kit following manufacturers recommendations and provided to the University of Alaska Fairbanks DNA Core Lab for 48-fold multiplexing using the Nextera XT DNA Library Preparation Kit and paired-end 300bp sequencing on the Illumina MiSeq. MiSeq runs in 2017—2019 yielded [] Gbp (51 genomes), 14.9 Gbp (35 genomes), and 13.8 Gbp (48 genomes), respectively. Raw reads were demultiplexed on-board the Illumina MiSeq, then adapter-trimmed using bbduk from the BBTools package (v38.44; parameters: ref=adapters,phix ktrim=r k=23 mink=11 hdist=1 tpe tbo) and assembled with SPAdes v3.13.0 using default parameters. Assembly graphs were visualized in Bandage v0.8.0 and manually edited to remove low-coverage and contaminant contigs. Quality-controlled assemblies were submitted to PATRIC v3.5.34 for annotation using RAST. Preliminary taxonomic assignments were made using sendsketch (BBtools; parameters: address=nt, or address=silva). Final taxonomic assignments were made using conserved core gene phylogenetic trees constructed in PATRIC. The raw reads can be accessed using SRA accessions XXXXXX. Genome assemblies can be accessed at accessions YYYYYY. Genome assembly graphs in .gfa format are available at http://github.com/rec3141/biol342. References Bushnell B. 2019. BBTools. http://sourceforge.net/projects/bbmap Nurk S, Bankevich A, Antipov D, Gurevich A, Korobeynikov A, Lapidus A, Prjibelsky A, Pyshkin A, Sirotkin A, Sirotkin Y, Stepanauskas R, McLean J, Lasken R, Clingenpeel SR, Woyke T, Tesler G, Alekseyev MA, Pevzner PA. 2013. Assembling genomes and mini-metagenomes from highly chimeric reads. In Deng M, Jiang R, Sun F, Zhang X (ed), RECOMB 2013: Research in Computational Molecular Biology. Springer, Berlin. Wattam AR, Davis JJ, Assaf R, Boisvert S, Brettin T, Bun C, Conrad N, Dietrich EM, Disz T, Gabbard JL, Gerdes S, Henry CS, Kenyon RW, Machi D, Mao C, Nordberg EK, Olsen GJ, Murphy-Olson DE, Olson R, Overbeek R, Parrello B, Pusch GD, Shukla M, Vonstein V, Warren A, Xia F, Yoo H, Stevens RL. 2017. Improvements to PATRIC, the all-bacterial Bioinformatics Database and Analysis Resource Center. Nucleic Acids Res 45:D535-D542. Wick RR, Schultz MB, Zobel J, Holt KE. 2015. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics 31:3350-3352. Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, Olson R, Overbeek R, Parrello B, Pusch GD, Shukla M, Thomason JA, Stevens R, Vonstein V, Wattam AR, Xia F. 2015. RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep 5:8365.