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02-01-ExperimentalPlanning.Rmd

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## What Is RNAseq
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RNAseq is a next generation sequencing technique for profiling all or selected target RNA molecules in a tissue from an organism. It typically involves isolating RNA, converting to cDNA, ligating adapter sequences to the cDNA then amplifying by PCR to construct a library that can be used for sequencing. The reads generated by sequencing are typically aligned to a reference genome.
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RNAseq is a next generation sequencing technique for profiling all or selected RNA molecules in a tissue from an organism. It typically involves isolating RNA, converting to cDNA, ligating adapter sequences to the cDNA then amplifying by PCR to construct a library that can be used for sequencing. The reads generated by a sequencing machine are then aligned to a reference genome and can then be used for analysis.
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A diverse ecosystem of protocols and technology exist that can be used to generate RNAseq data, which can be used in a wide variety of applications.
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(ref:foo1) Overview of the steps in an RNAseq experiment. At each of these steps, there are choices that are made that can influence the final output of the experiment. Image source: [RNA Sequencing Data: Hitchhiker's Guide to Expression Analysis, 2019](https://www.annualreviews.org/content/journals/10.1146/annurev-biodatasci-072018-021255)
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### What is a sequencing machine doing? {- .rationale}
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A sequencing machine generates a readout of the nucleotides that make up the original RNA molecule, typically by using uniquely labelled flourescent nucleotides. Every time one of these nucleotides are added to a strand of DNA, the fluorescence emitted is detected by the sequencing machine.
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A sequencing machine generates a readout of the nucleotides that make up a DNA fragement. The most common type of sequencing is Sequencing by Synthesis which involves the use of unique fluorescently labelled nucleotides. These nucleotides are added one at a time and every time one of these nucleotides are added to a strand of DNA, the fluorescence emitted is detected by the sequencing machine.
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(ref:foo2) Illumina Sequencing by Synthesis. Image source: [Microbenotes: Illumina Sequencing](https://microbenotes.com/illumina-sequencing/)
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```{r, echo=FALSE, out.width="90%", fig.cap="(ref:foo2)"}
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knitr::include_graphics("images/experimental_design/fig_illumina_sequencing.png")
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```
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##
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A diverse ecosystem of protocols and technology exist that can be used to generate RNAseq data, which can be used in a wide variety of applications.
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## Planning an RNAseq Experiment

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