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100644 images/Illumina_SBS.019.png delete mode 100644 images/quasi-mapping_overview.png diff --git a/Markdowns/01_Introduction_to_RNAseq_Methods.Rmd b/Markdowns/01_Introduction_to_RNAseq_Methods.Rmd index 2ba9a74..992d5d9 100644 --- a/Markdowns/01_Introduction_to_RNAseq_Methods.Rmd +++ b/Markdowns/01_Introduction_to_RNAseq_Methods.Rmd @@ -1,6 +1,6 @@ --- title: "Introduction to RNAseq Methods" -date: "March 2023" +date: "October 2024" output: ioslides_presentation: css: css/stylesheet.css @@ -93,7 +93,7 @@ output:
-**Experimental Design** +**Experimental Design**
@@ -112,61 +112,23 @@ output:
- +

Image adapted from: Wang, Z., et al. (2009), Nature Reviews Genetics, 10, 57–63.

- - - - - - - - - - -## Designing the right experiment - - - - - - - - - -### A good experiment should: - -* Have clear objectives - -* Have sufficient power - -* Be amenable to statisical analysis - -* Be reproducible - -* More on experimental design later - - -## Designing the right experiment - -### Practical considerations for RNAseq - +## Practical considerations for RNAseq + * Coverage: how many reads? - + * Read length & structure: Long or short reads? Paired or Single end? - -* Controlling for batch effects - + * Library preparation method: Poly-A, Ribominus, other? - -## Designing the right experiment - How many reads do we need? - - + +## How many reads do we need? +

The coverage is defined as: @@ -176,191 +138,24 @@ $\frac{Read\,Length\;\times\;Number\,of\,Reads}{Length\,of\,Target\,Sequence}$

-The amount of sequencing needed for a given sample is determined by the goals of -the experiment and the nature of the RNA sample. - - * For a general view of differential expression: 5–25 million reads per sample * For alternative splicing and lowly expressed genes: 30–60 million reads per sample. * In-depth view of the transcriptome/assemble new transcripts: 100–200 million reads * Targeted RNA expression requires fewer reads. * miRNA-Seq or Small RNA Analysis require even fewer reads. - + ## Designing the right experiment - Read length -### Long or short reads? Paired or Single end? +Long or short reads? Paired or Single end? The answer depends on the experiment: * Gene expression – typically just a short read e.g. 50/75 bp; SE or PE. * kmer-based quantification of Gene Expression (Salmon etc.) - benefits from PE. * Transcriptome Analysis – longer paired-end reads (such as 2 x 75 bp). -* Small RNA Analysis – short single read, e.f. SE50 - will need trimming. - - - - -## Designing the right experiment - Replication - -### Biological Replication - -* Measures the biological variations between individuals - -* Accounts for sampling bias - -### Technical Replication - -* Measures the variation in response quantification due to imprecision in the -technique - -* Accounts for technical noise - - -## Designing the right experiment - Replication - -### Biological Replication - -
-Each replicate is from an indepent biological individual - -* *In Vivo*: - - * Patients - * Mice - -* *In Vitro*: - - * Different cell lines - * Different passages +* Small RNA Analysis – short single read, e.g. SE50 - will need trimming. + -
- -
- -
- -## Designing the right experiment - Replication - -### Technical Replication - - -Replicates are from the same individual but processed separately - -* Experimental protocol -* Measurement platform - - -
- -
- -## Designing the right experiment - Batch effects - -* Batch effects are sub-groups of measurements that have qualitatively different behavior across conditions and are unrelated to the biological or scientific variables in a study. - -* Batch effects are problematic if they are confounded with the experimental variable. - -## Designing the right experiment - Batch effects - - - -## Designing the right experiment - Batch effects - - - -## Designing the right experiment - Batch effects - -* Batch effects are sub-groups of measurements that have qualitatively different behavior across conditions and are unrelated to the biological or scientific variables in a study. - -* Batch effects are problematic if they are confounded with the experimental variable. - -* Batch effects that are randomly distributed across experimental variables can be controlled for. - -## Designing the right experiment - Batch effects - -* Batch effects are sub-groups of measurements that have qualitatively different behavior across conditions and are unrelated to the biological or scientific variables in a study. - -* Batch effects are problematic if they are confounded with the experimental variable. - -* Batch effects that are randomly distributed across experimental variables can be controlled for. - -* Randomise all technical steps in data generation in order to avoid batch effects. - - - -## Designing the right experiment - Batch effects - -* Batch effects are sub-groups of measurements that have qualitatively different behavior across conditions and are unrelated to the biological or scientific variables in a study. - -* Batch effects are problematic if they are confounded with the experimental variable. - -* Batch effects that are randomly distributed across experimental variables can be controlled for. - -* Randomise all technical steps in data generation in order to avoid batch effects. - - - -## Designing the right experiment - Batch effects - -* Batch effects are sub-groups of measurements that have qualitatively different behavior across conditions and are unrelated to the biological or scientific variables in a study. - -* Batch effects are problematic if they are confounded with the experimental variable. - -* Batch effects that are randomly distributed across experimental variables can be controlled for. - -* Randomise all technical steps in data generation in order to avoid batch effects. - - - -## Designing the right experiment - Batch effects - -* Batch effects are sub-groups of measurements that have qualitatively different behavior across conditions and are unrelated to the biological or scientific variables in a study. - -* Batch effects are problematic if they are confounded with the experimental variable. - -* Batch effects that are randomly distributed across experimental variables can be controlled for. - -* Randomise all technical steps in data generation in order to avoid batch effects - -* **Record everything**: Age, sex, litter, cell passage .. - - -## RNAseq Workflow - - -
- - -
-**Experimental Design** -
- -
-**Library Preparation** -
- -
-**Sequencing** -
- -
-**Bioinformatics Analysis** -
-
- -
- - -
- -
-

Image adapted from: Wang, Z., et al. (2009), Nature Reviews Genetics, 10, 57–63.

-
## Library preparation @@ -372,7 +167,7 @@ Replicates are from the same individual but processed separately position: absolute; top: 0px; left: 0px"> - +
- - Ribosomal RNA + - Ribosomal RNA
- - Poly-A transcripts + - Poly-A transcripts
- - Other RNAs e.g. tRNA, miRNA etc. + - Other RNAs e.g. tRNA, miRNA etc.
@@ -406,7 +201,7 @@ Replicates are from the same individual but processed separately
- +
Poly-A transcripts e.g.: @@ -424,7 +219,7 @@ Poly-A transcripts e.g.:
- +
Poly-A transcripts + Other mRNAs e.g.: @@ -435,43 +230,9 @@ Poly-A transcripts + Other mRNAs e.g.:
-## RNAseq Workflow - - -
- - -
-**Experimental Design** -
- -
-**Library Preparation** -
- -
-**Sequencing** -
- -
-**Bioinformatics Analysis** -
-
- -
- - -
- -
-

Image adapted from: Wang, Z., et al. (2009), Nature Reviews Genetics, 10, 57–63.

-
- -## Sequencing by synthesis +## Sequencing by Synthesis -A complimentary strand is synthesized using the cDNA fragment as template. +A complimentary strand is synthesized using the cDNA fragment as template. Each nucleotide includes a fluorescent tag and as the new strand is synthesized, the colour of the fluorescence indicates which base is being added. @@ -479,98 +240,26 @@ the colour of the fluorescence indicates which base is being added. The sequencer records the order of these flashes of light and translates them to a base sequence. -[see this animation](https://emea.illumina.com/science/technology/next-generation-sequencing/sequencing-technology.html) - -
- -
- -
- -
- -## Sequencing by synthesis - sequencing errors Sequencing errors cause uncertainty in calling the nucleotide at a given -location. These reductions in confidence would be reflected int he quality +location. These reductions in confidence would be reflected in the quality scores in your fastq output. -
- -If a probe doesn't shine as bright as it should, the sequencer is less confident -in calling that base. - - - - -
- -
- -If there are lots of probes the same colour in the same region the sequencer -finds it harder to identify the individual reads. - - - -
- -## RNAseq Workflow - - -
- - -
-**Experimental Design** -
- -
-**Library Preparation** -
- -
-**Sequencing** -
- -
-**Bioinformatics Analysis** -
+
+
-
- - +
+
-
-

Image adapted from: Wang, Z., et al. (2009), Nature Reviews Genetics, 10, 57–63.

-
## Case Study - + ## Differential Gene Expression Analysis Workflow {#less_space_after_title}

- + diff --git a/Markdowns/01_Introduction_to_RNAseq_Methods.html b/Markdowns/01_Introduction_to_RNAseq_Methods.html index 1c7f7ed..e44e3af 100644 --- a/Markdowns/01_Introduction_to_RNAseq_Methods.html +++ b/Markdowns/01_Introduction_to_RNAseq_Methods.html @@ -3061,20 +3061,23 @@ }); }; -if (document.readyState !== "loading" && - document.querySelector('slides') === null) { - // if the document is done loading but our element hasn't yet appeared, defer - // loading of the deck - window.setTimeout(function() { - loadDeck(null); - }, 0); -} else { - // still loading the DOM, so wait until it's finished - document.addEventListener("DOMContentLoaded", loadDeck); +if (!window.Shiny) { + // If Shiny is loaded, the slide deck is initialized in ioslides template + + if (document.readyState !== "loading" && + document.querySelector('slides') === null) { + // if the document is done loading but our element hasn't yet appeared, defer + // loading of the deck + window.setTimeout(function() { + loadDeck(null); + }, 0); + } else { + // still loading the DOM, so wait until it's finished + document.addEventListener("DOMContentLoaded", loadDeck); + } } - + + + + + + + + + + + + + + + + + + + + + + + +
+

+ +

+

October 2024

+
+
+ +

Differential Gene Expression Analysis Workflow

+ +
+
+ +

+ +

Transformation

+ +

For differential expression analyses we use raw counts but to visualise the data to explore it we use transformed data.

+ +
+
+

+ +
+
    +
  • The range of raw counts is very large
  • +
  • Variance increases with mean gene expression
  • +
+ +
+
+

+ +
+
    +
  • Allows us to more clearly assess differences between sample groups
  • +
+ +

Types of Transformations

+ +
    +
  • Log2
  • +
  • Rlog - Performs a log2 scale transformation in a way that compensates for differences between samples for genes with low read count and also normalizes between samples for library size.
  • +
  • VST - Variance stabilizing transformation (VST) aims at generating a matrix of values for which variance is constant across the range of mean values, especially for low mean and accounts for library size.
  • +
+ +

Comparison between the two: https://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#count-data-transformations

+ +

Principle Component Analysis

+ +
    +
  • Unsupervised analysis
  • +
  • If the experiment is well controlled and has worked well, we should find that replicate samples cluster closely, whilst the greatest sources of variation in the data should be between treatments/sample groups
  • +
  • Useful tool for checking for outliers and batch effects
  • +
+ +

+ + + + +
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