II Choose Working Directory Mitogenome Assembly from NGS Genome Skimming Data

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Mitogenome Assembly from NGS Genome Skimming Data: II. Choose Working Directory by Avery S. Hiley* • *Corresponding author: • Avery S. Hiley ([email protected] | [email protected]) • Graduate Student Researcher, Rouse Lab • Scripps Institution of Oceanography, UC San Diego • http://www.spineless.info/avery-hiley... • / avery-sea • Description: • This protocol provides thorough instructions for how to assemble and annotate full mitochondrial genomes from NGS genome skimming data (specifically paired-end, FASTQ, gzipped reads). The steps are categorized in the following sections, which include detailed explanations throughout (e.g. why specific steps are executed in the manner instructed): • I. Download Programs and Dependencies: • I. Download Programs and Dependencies... • II. Choose Working Directory: • II. Choose Working Directory [Mitogen... • III. Simple Stats with SeqKit: • III. Simple Stats with SeqKit [Mitoge... • IV. Clean and Trim Reads with Trimmomatic: • IV. Clean and Trim Reads with Trimmom... • V. Downsample Reads with MITObim: • V. Downsample Reads with MITObim [Mit... • VI. Reformat Reads to Non-Interleaved with BBMap: • VI. Reformat Reads to Non-Interleaved... • VII. Mitogenome Assembly and Annotation: MitoFinder with MetaSPAdes: • VII. Mitogenome Assembly and Annotati... • VIII. Mitogenome Assembly with NOVOPlasty: • VIII. Mitogenome Assembly with NOVOPl... • Furthermore, there are supplementary tutorial videos (YouTube links provided above) for each of the aforementioned protocol sections, which show all of the corresponding steps being executed. These videos may be used for extra guidance while running the protocol, and are especially helpful to see how long the commands take to execute and successfully terminate in real-time with real NGS genome skimming data. • Guidelines: • The current version of this protocol is streamlined for Mac operating systems. However, alterations to the protocol for other operating systems (e.g. Windows OS and Linux) should be relatively simple, mainly requiring the use of different apps with equivalent applications to the macOS apps specified (e.g. Terminal and Finder), and different download links for installation of the required programs and dependencies. • Before Start: • Download the NGS genome skimming data (paired-end, FASTQ, gzipped reads) for the species of interest. There should be 2 files total per gDNA extraction sequenced, typically ending in 1.fq.gz and 2.fq.gz if the data was obtained from Novogene. Additionally, you must have a COI Sanger Sequence (FASTA format) obtained from the exact gDNA extraction of the species/sample of interest if you perform Section VIII. Mitogenome Assembly with NOVOPlasty. • Materials: • Detailed instructions on how to correctly download the required programs and their corresponding dependencies are provided in Section I. Download Programs and Dependencies, along with links and citations. Therefore, it is not necessary to download the following programs beforehand; they are merely listed here for your reference. • 1. Xcode Version 12.1 • 2. Homebrew • 3. SeqKit v0.13.2 • 4. Trimmomatic Version 0.39 • 5. MITObim Version 1.9.1 • 6. BBMap Version 38.87 • 7. MitoFinder v1.4 • 8. NOVOPlasty Version 4.2

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