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# Introduction to Bulk RNA-seq data analysis
### 17th, 24th Nov and 1st Dec
#### Online
### 15, 18, 19 March 2024
#### In person
#### Bioinformatics Training Facility, Craik-Marshall Building, Downing Site, University of Cambridge

![](Bulk_RNAseq_Course_Base/images/CRUK_Cambridge_Major_Centre_logo.jpg)

## Instructors

* Abigail Edwards - Bioinformatics Core, Cancer Research UK Cambridge Institute
* Adam Reid - Gurdon Institute, University of Cambridge
* Tom Smith - MRC Toxicology, University of Cambridge
* Ulrika Yuan - Department of Biochemistry, University of Cambridge
* Zac Scurlock - Department of Pathology, University of Cambridge
* Ash Sawle - Bioinformatics Core, Cancer Research UK Cambridge Institute
* Chandra Chilamakuri - Bioinformatics Core, Cancer Research UK Cambridge Institute
* Erin Doody - Sainsbury Laboratory, University of Cambridge
* Ian Tsang - NIAB, Cambridge
* Puspendu Sardar - Department of Medicine, University of Cambridge


## Outline
Expand All @@ -33,127 +33,90 @@ be aware that timings for different sections laid out in the schedule below may
not be adhered to. There may be some necessity to make adjustments to the course
as we go.

> ## Prerequisites
>
> __**Some basic experience of using a UNIX/LINUX command line is assumed**__
>
> __**Some R knowledge is assumed and essential. Without it, you will struggle on this course.**__
> If you are not familiar with the R statistical programming language we
> strongly encourage you to work through an introductory R course before
> attempting these materials.
> We recommend our [Introduction to R course](https://bioinformatics-core-shared-training.github.io/r-intro/)
## Prerequisites

## Shared Google Document
* __**Basic experience of using a UNIX/LINUX command line is assumed**__
* Watch this [overview video](https://drive.google.com/file/d/1qo_eNXaYSphU_Xpk0jzDHp1wJzyfSwAT/view?usp=drive_link) (and [accompanying materials](https://cambiotraining.github.io/unix-shell/) up to section 6).

This [Google Document](https://docs.google.com/document/d/1nqnelPJFx_Gvw8Mz83AEzcP2cII2ZlLpMlRDBEdWFeU/edit?usp=sharing) contains useful information and links.

Please use it to post any long form questions you have during the course.

The trainers will be monitoring the document and will answer questions as quickly as they can.

## Introduce Yourself

There is another Google Doc [Google Document](https://docs.google.com/document/d/19xeAvseME9N7x0NE8hMOm3MUCHxKg0IiHhSQuc0fBDE/edit?usp=drive_link).
Please write a couple sentences here to introduce yourself to the class, tell us a bit about your background and what you hope to get out of this course.
If you are a student or staff at the University of Cambridge, tell us which Department you are in.

## Course etiquette

As this course is being taught online and there are a large number of participants,
we will all need to follow a [few simple rules](https://docs.google.com/presentation/d/e/2PACX-1vQv9nTlsdRC9iZJU138tLL1jrwNoryp8P-FnXxb_ugOOWjbav4QHTLYLLZj2KK4kTO0_3x3VlzSdrUu/pub?start=false&loop=false&delayms=3000) to ensure things run as smoothly as possible:

1. Please mute your microphone

2. To get help from a tutor, please click the "Raise Hand" button in Zoom:

![](Bulk_RNAseq_Course_Base/images/raise_hand.png)

This can be found by clicking on the 'Participants' button. A tutor will
then contact you in the chat. If necessary, you and the tutor can be moved
to a breakout room where you can discuss your issue in more detail.

3. Please ask any general question by typing it into the Google Doc mentioned above

4. During practicals, when you are done, please press the green "Yes" button:

![](Bulk_RNAseq_Course_Base/images/yes_button.png)

This way we will know when we can move on.
* __**Some R knowledge is assumed and essential. Without it, you will struggle on this course.**__
* If you are not familiar with the R statistical programming language we strongly encourage you to work through an introductory R course before attempting these materials.
* We recommend our [Introduction to R course](https://bioinformatics-core-shared-training.github.io/r-intro/)


## Timetable

### Day 1

**Trainers:** Abbi, Adam, Ulrika, Tom, Zac
**Trainers:** Ash, Chandra, Erin, Ian, Puspendu

9:30 - 9:45 - [Welcome](https://docs.google.com/presentation/d/1u-UFD5LzBh0FTV54e-TCJJOrCRtdWxM000rQw-a5w6w/edit?usp=sharing)! <!-- Hugo/Paul/Abbi -->
9:30 - 9:45 - [Welcome](https://docs.google.com/presentation/d/1u-UFD5LzBh0FTV54e-TCJJOrCRtdWxM000rQw-a5w6w/edit?usp=sharing)! <!-- Hugo/Paul -->

9:45 - 10:15 - [Introduction to RNAseq Methods](Bulk_RNAseq_Course_Base/Markdowns/01_Introduction_to_RNAseq_Methods.html) - Tom
9:45 - 10:15 - [Introduction to RNAseq Methods](Bulk_RNAseq_Course_Base/Markdowns/01_Introduction_to_RNAseq_Methods.html) - Erin

10:15 - 11:00 [Raw read file format and QC](Bulk_RNAseq_Course_Base/Markdowns/02_FastQC_introduction.html) - Adam
10:15 - 11:00 [Raw read file format and QC](Bulk_RNAseq_Course_Base/Markdowns/02_FastQC_introduction.html) - Ian

- [Practical](Bulk_RNAseq_Course_Base/Markdowns/02_FastQC_practical.html)
<!-- - [Practical solutions](Bulk_RNAseq_Course_Base/Markdowns/02_FastQC_practical.Solutions.html) --->
<!-- - [Practical solutions](Bulk_RNAseq_Course_Base/Markdowns/02_FastQC_practical.Solutions.html) -->

11:00 - 13:30 [Alignment and Quantification of Gene Expression with Salmon](Bulk_RNAseq_Course_Base/Markdowns/03_Quantification_with_Salmon_introduction.html) - Adam
11:00 - 13:30 [Alignment and Quantification of Gene Expression with Salmon](Bulk_RNAseq_Course_Base/Markdowns/03_Quantification_with_Salmon_introduction.html) - Chandra

- [Practical](Bulk_RNAseq_Course_Base/Markdowns/03_Quantification_with_Salmon_practical.html)
- [Practical solutions](Bulk_RNAseq_Course_Base/Markdowns/03_Quantification_with_Salmon_practical.Solutions.html)
<!-- - [Practical solutions](Bulk_RNAseq_Course_Base/Markdowns/03_Quantification_with_Salmon_practical.Solutions.html) -->

13:30 - 14:30 Lunch

14:30 - 15:30 [QC of alignment](Bulk_RNAseq_Course_Base/Markdowns/04_Quality_Control_introduction.html) - Ulrika
14:30 - 15:30 [QC of alignment](Bulk_RNAseq_Course_Base/Markdowns/04_Quality_Control_introduction.html) - Chandra

- [Practical](Bulk_RNAseq_Course_Base/Markdowns/04_Quality_Control_practical.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/04_Quality_Control_practical.pdf))
- [Practical solutions](Bulk_RNAseq_Course_Base/Markdowns/04_Quality_COntrol_practical.Solutions.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/04_Quality_Control_practical.Solutions.pdf))
<!-- - [Practical solutions](Bulk_RNAseq_Course_Base/Markdowns/04_Quality_COntrol_practical.Solutions.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/04_Quality_Control_practical.Solutions.pdf)) -->

15.30 - 17.30 [Data Exploration in R](Bulk_RNAseq_Course_Base/Markdowns/05_Data_Exploration.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/05_Data_Exploration.pdf)) - Abbi
15.30 - 17.30 [Data Exploration in R](Bulk_RNAseq_Course_Base/Markdowns/05_Data_Exploration.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/05_Data_Exploration.pdf)) - Ash

- [Practical solutions](Bulk_RNAseq_Course_Base/Markdowns/05_Data_Exploration.Solutions.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/05_Data_Exploration.Solutions.pdf))
- [Live Script](live_scripts/data_exploration.R)
<!-- - [Practical solutions](Bulk_RNAseq_Course_Base/Markdowns/05_Data_Exploration.Solutions.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/05_Data_Exploration.Solutions.pdf)) -->
<!-- - [Live Script](live_scripts/data_exploration.R) -->


### Day 2

**Trainers:** Abbi, Adam, Raquel, Tom
**Trainers:** Ash, Chandra, Erin, Ian, Puspendu

<!-- Welcome Announcements - Abbi -->
9:30 - 10:15 [Introduction to RNAseq Analysis in R](Bulk_RNAseq_Course_Base/Markdowns/06_Introduction_to_RNAseq_Analysis_in_R.html) - Abbi
9:30 - 10:15 [Introduction to RNAseq Analysis in R](Bulk_RNAseq_Course_Base/Markdowns/06_Introduction_to_RNAseq_Analysis_in_R.html) - Ash

10:15 - 13:00 Statistical Analysis of Bulk RNAseq Data
10:15 - 13:00 Statistical Analysis of Bulk RNAseq Data - Ash

- Part I: [Statistics of RNA-seq analysis](Bulk_RNAseq_Course_Base/additional_scripts_and_materials/RNA-seq_stats.pdf) - Abbi
- Part II: [Linear Models in R and DESeq2](Bulk_RNAseq_Course_Base/Markdowns/07_Linear_Models.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/07_Linear_Models.pdf)) - Abbi
- Part I: [Statistics of RNA-seq analysis](Bulk_RNAseq_Course_Base/additional_scripts_and_materials/RNA-seq_stats.pdf)
- Part II: [Linear Models in R and DESeq2](Bulk_RNAseq_Course_Base/Markdowns/07_Linear_Models.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/07_Linear_Models.pdf))
- [Slides](Bulk_RNAseq_Course_Base/additional_scripts_and_materials/Statistical_models_in_R_DESeq2.pdf)
- Find the worksheet in `Course_Materials/stats/models_in_r_worksheet.R`

13:00 - 14:00 Lunch

14:00 - 17:30 - [Differential Expression for RNA-seq](Bulk_RNAseq_Course_Base/Markdowns/08_DE_analysis_with_DESeq2.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/08_DE_analysis_with_DESeq2.pdf)) - Adam
- [practical solutions](Bulk_RNAseq_Course_Base/Markdowns/08_DE_analysis_with_DESeq2.Solutions.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/08_DE_analysis_with_DESeq2.Solutions.pdf))
- [live script](live_scripts/deseq_day2_afternoon.R) -->
14:00 - 17:30 - [Differential Expression for RNA-seq](Bulk_RNAseq_Course_Base/Markdowns/08_DE_analysis_with_DESeq2.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/08_DE_analysis_with_DESeq2.pdf)) - Chandra

<!-- - [practical solutions](Bulk_RNAseq_Course_Base/Markdowns/08_DE_analysis_with_DESeq2.Solutions.html) ([pdf](Bulk_RNAseq_Course_Base/Markdowns/08_DE_analysis_with_DESeq2.Solutions.pdf)) -->
- [DESeq2 results extraction cheatsheet](Bulk_RNAseq_Course_Base/additional_scripts_and_materials/DESeq2_results_cheatsheet.pdf)
- [extra models plots](Bulk_RNAseq_Course_Base/additional_scripts_and_materials/Expl_all.pdf)

### Day 3

**Trainers:** Abbi, Adam, Ulrika, Raquel
**Trainers:** Ash, Chandra, Erin, Ian, Puspendu

9.30 - 9.45 - [Recap of Day 1 and 2](Bulk_RNAseq_Course_Base/additional_scripts_and_materials/Analysis_of_RNA-seq_data_day3recap.pdf) - Abbi
9.30 - 9.45 - [Recap of Day 1 and 2](Bulk_RNAseq_Course_Base/additional_scripts_and_materials/Analysis_of_RNA-seq_data_day3recap.pdf) - Chandra

9.45 - 12.30 [Annotation and Visualisation of RNA-seq results](Bulk_RNAseq_Course_Base/Markdowns/09_Annotation_and_Visualisation.html) - Ulrika
- [practical solutions](Bulk_RNAseq_Course_Base/Markdowns/09_Annotation_and_Visualisation_Solutions.html)
- [live script](live_scripts/Live_script_Ulrika.R)
9.45 - 12.30 [Annotation and Visualisation of RNA-seq results](Bulk_RNAseq_Course_Base/Markdowns/09_Annotation_and_Visualisation.html) - Ian / Chandra
<!-- - [practical solutions](Bulk_RNAseq_Course_Base/Markdowns/09_Annotation_and_Visualisation_Solutions.html) -->
<!-- - [live script](live_scripts/Live_script_Ulrika.R) -->

12.30 - 13.30 Lunch

13.30 - 16:30 [Gene-set testing](Bulk_RNAseq_Course_Base/Markdowns/10_Gene_set_testing_introduction.html) - Abbi
13.30 - 16:30 [Gene-set testing](Bulk_RNAseq_Course_Base/Markdowns/10_Gene_set_testing_introduction.html) - Ash

- [Practical (html)](Bulk_RNAseq_Course_Base/Markdowns/10_Gene_set_testing.html) [(rmd)](Bulk_RNAseq_Course_Base/Markdowns/10_Gene_set_testing.Rmd) [(pdf)](Bulk_RNAseq_Course_Base/Markdowns/10_Gene_set_testing.pdf)
- [Practical solutions (html)](Bulk_RNAseq_Course_Base/Markdowns/10_Gene_set_testing.Solutions.html) [(rmd)](Bulk_RNAseq_Course_Base/Markdowns/10_Gene_set_testing.Solutions.Rmd) [(pdf)](Bulk_RNAseq_Course_Base/Markdowns/10_Gene_set_testing.Solutions.pdf)
- [Live Script](live_scripts/GeneSetTesting.R)
<!-- - [Practical solutions (html)](Bulk_RNAseq_Course_Base/Markdowns/10_Gene_set_testing.Solutions.html) [(rmd)](Bulk_RNAseq_Course_Base/Markdowns/10_Gene_set_testing.Solutions.Rmd) [(pdf)](Bulk_RNAseq_Course_Base/Markdowns/10_Gene_set_testing.Solutions.pdf) -->
<!-- - [Live Script](live_scripts/GeneSetTesting.R) -->

<!-- Goodbye: Abbi -->
<!-- Goodbye: Ash -->

## Data, software and materials

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