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Nanopore RNA Sequencing Protocol
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=Introduction= Hello, and welcome to the mRNA m6A modification detection guide and protocol. This protocol is a fully comprehensive guide designed to help you get a Linux (or compatible shell) computer up and running from scratch for m6A mRNA modification detection using direct Nanopore sequencing. The initial goal of this project was to detect m6A mRNA modifications in glioma samples, the coveted '''g'''lioma m'''R'''NA m6'''A''' '''M'''ethylator '''P'''henotype (or “'''G-RAMP'''”), analogous to epigenetic modifications at CpG sites in glioma ('''g'''lioma '''C'''pG '''I'''sland '''M'''ethylator '''P'''henotype, or “'''G-CIMP'''”). However, this protocol can be used to detect any m6A mRNA modifications in a sample, with a few minor modifications. With all of this in mind, I have written this protocol in as a generic way as possible, describing everything I can along the way, so that hopefully anyone reading this can replicate what I’ve done without any R knowledge or computer programming experience. Some of the code may be intimidating at times, but I will try to explain each syntax as I go along so that you can understand what each line is doing as you move through the code. This protocol is separated into six major sections: Introduction, Requirements (and Installations, if necessary), Running Samples (wet lab portion), Sequencing, m6A Detection (via two different methods), and Final Analysis. Each of these sections utilize different skills and/or software for processing samples and data (for example, sequencing and alignment will be handled via Guppy and minimap2, while m6A detection will be handled via m6anet), which will be outlined at the start of the protocol. Where applicable, I will include '''warning''' boxes to help you avoid running into errors, and '''''should never be glossed over''''' when doing a run. Other bits of information that are not as paramount to a successful run will be included in block quotes. At the end of the protocol, I will also include an Appendix section, which will contain technical terms that will be used throughout the guide and an Additional Information section containing useful information about the nanopore system itself. Finally, a big shoutout to '''Bryan Kevan''' and '''Sean Pianka''', who laid down the ground work for this project, '''Albert Lai''' for his support and encouragement, '''Luca Cozzuto''' and the '''Goeke lab''' for their help in troubleshooting at challenging steps of getting the pipeline up to speed, and the latter for the development of ''m6anet''. Without them, this project would not have been possible. Without further adieu, let’s get to it!
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