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Nanopore RNA Sequencing Protocol
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=Appendix= * '''Alignment:''' A process by which a given DNA or RNA sequence is matched with a reference genome / transcriptome, and therefore given a gene name. * '''Bam:''' A type of DNA or RNA sequencing file containing your reads after they have been mapped to the genome. It is a binary version of the earlier .SAM file, which is where it gets its name. * '''Bam.bai:''' An index file with the same prefix name as a .bam file. This file acts like an external table of contents, which allows algorithms and software to be able to jump to specfic sections of the .bam file without having to read through all the sequences. * '''Basecalling:''' A term used to refer to the process of obtaining direct measurements of ionic current as DNA/RNA is fed through a nanopore, and then using that information to obtain DNA/RNA nucleotide “reads”. Basecalling is achieved by running the raw Fast5 files through the Guppy software. * '''Conda:''' A collection of tools and runtime environment for installing software on a home machine. * '''CUDA:''' Stands for “Compute Unified Device Architecture”, which allows GPU resources to be used for general processing. Developed by and for NVIDIA GPUs. * '''Docker:''' A Linux container whereby you can run Linux code and commands in a virtual environment. I originally used this with an earlier version of this protocol, but it may not be required anymore. Alternative to singularity. * '''Fast5:''' A type of raw file obtained from sequencing DNA or RNA molecules. This can be converted into a Fastq file using the Guppy software provided by Oxford Nanopore. * '''Fastq:''' A type of raw file obtained from sequencing DNA or RNA molecules and houses the sequenced genetic material. Fastq files are often a processed step up from Fast5 files, since they contain some metadata about the sequenced material. * '''Flow cell:''' A term used for the sheet that contains the nanopores that sequence DNA/RNA data, along with their electrodes and sensor chip. * '''Guppy:''' Guppy is a basecalling algorithm provided by Oxford Nanopore Technologies, which reads the fast5 or fastq files obtained from the Oxford Nanopore (such as “MinION”), and generates the DNA/RNA bases. * '''MinION / GridION:''' Two different hardware enclosures that house ONT flow cells while sequencing is occurring and relays that information to a computer. * '''Nanopore:''' Microscopic protein pores embedded in an electrically-resistant polymer membrane and used to process DNA/mRNA strands. * '''Nuclease-free Water:''' Water that has been purified to not be contaminated by non-sample nucleases. This is important for gene sequencing and PCR protocols, to ensure that the material you are working with is only from your sample. * '''ONT:''' A reference to “'''O'''xford '''N'''anopore '''T'''echnologies”, the manufacturer of the Nanopore we use in the lab. You will see this in a lot of folders that house files from the company, such as “ont-guppy”, which is used to sequence raw nanopore data. * '''Python:''' A type of programming language. * '''R:''' Programming language commonly used by scientists to analyze data. * '''Reads:''' Term used to refer to sequenced genetic material (DNA or RNA). * '''Root Folder:''' The parent folder location that houses most of your files. For the purposes of these guides, we are considering the root folder at <code>/home/username/</code> (Linux) or <code>/Users/username</code> (Mac). Denoted by “root” or <code>~</code>. * '''.SAM:''' A type of file format that holds processed sequencing data. Stands for “'''S'''equence '''A'''lignment '''M'''ap format”. * '''Shell Script:''' A series of code that can be executed at once via the shell (i.e. in ''Terminal''). * '''Singularity:''' A Linux container whereby you can run Linux code and commands in a virtual environment. Alternative to docker. * '''Terminal:''' An application on both Linux and Mac machines that gives you access to the shell whereby you can execute commands. * '''XCode:''' Integrative Development Environment and collection of programming tools provided by Apple, Inc. for developing software.
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