diff --git a/_site/omics/week-3/images/88H-exp-design-jillian.png b/_site/omics/week-3/images/88H-exp-design-jillian.png new file mode 100644 index 0000000..e46a2ba Binary files /dev/null and b/_site/omics/week-3/images/88H-exp-design-jillian.png differ diff --git a/_site/omics/week-3/study_before_workshop.html b/_site/omics/week-3/study_before_workshop.html index 6b4a1ac..bc51bab 100644 --- a/_site/omics/week-3/study_before_workshop.html +++ b/_site/omics/week-3/study_before_workshop.html @@ -1,898 +1,887 @@ - - - - - - - - - -Data Analysis for Group Project - Independent Study to prepare for workshop - - - - - + - - - - - + - - - - - - - - - - + + + - + + Data Analysis for Group Project - Independent Study to prepare for workshop + + + + + + + + + + + + + + + + + +
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Independent Study to prepare for workshop

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Omics 1: Hello data!

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+Emma Rand +
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6 October, 2023

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Sequence Data

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  • reads
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  • quality control
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  • align/pseudoalign
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  • quantify
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  • normalise
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🚧 NB still in construction 🚧

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Aims

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Overview

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  • find important genes
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  • what is ‘important’ -
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    • expressed
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    • differentially expressed i.e., which genes are significantly higher in one group vs another
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  • Concise summary of the experimental design and aims

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  • What the raw data consist of

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  • What has been done to the data so far

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  • What steps we will take in the workshop

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What is a read

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The Data

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There are three datasets

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  • FASTQ format files -
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    • sequences and information about each sequence’s read accuracy
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    • +

  • 🐸 transcriptomic data (bulk RNA-seq) from frog embryos.

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  • 🐭 transcriptomic data (single cell RNA-seq) from stemcells

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  • 🍂 ??????? Metabolomic / Metagenomic data from anaerobic digesters

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Differential expression

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  • what is differentially expressed
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  • how are DE genes related
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  • what are DE genes involved with
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Experimental design

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Stem cells: background

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Cells were sorted using flow cytometry on the basis of cell surface markers. There are three cell types:

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🐸 Experimental design

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Schematic of frog development experiment

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🐸 Experimental design

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Schematic of frog development experiment

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  • long-term haematopoetic stem cells (LT-HSCs) defined as : Lineage- ckit+ Sca1+ CD34- Flk2-

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  • haematopoetic stem and progenitor cells (HSPCs) defined as : Lineage- Sca1+ ckit+

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  • progenitor cells (Progs) defined as : Lineage- Sca1- ckit+

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  • 3 fertilisations

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  • two siblings from each fertilisation one control, on FGF treated

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  • sequenced at three time points

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  • 3 x 2 x 3 = 18 groups

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Each cell is then sequenced to quantify all the transcripts in each cell. different transcripts (genes) were identified.

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Stem cells: background

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🐸 Experimental design

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Schematic of frog development experiment

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Stem cells: Processing so far

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🐸 Aim

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  • selection of cells
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  • selection of genes: subset of surfaceome
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  • log2 normalised values
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  • find genes important in frog development

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  • Important means genes that are differentially expressed between the control and the FGF treated sibling

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  • Differentially expressed means the expression on one group is signifcantly higher than the other

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Stem cells: Aims

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🐸 Guided analysis

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  • Find interesting cell surface molecule genes that vary between cell types.
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  • The workshops will take you through comparing the control and FGF treated sibling at S30

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  • This is the “least interesting” comparison

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  • You will be guided to carefully document your work so you can apply the same methods to other comparisons

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Frog development: background

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🐭 Experimental design

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Schematic of stem cell experiment

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🐭 Experimental design

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Schematic of stem cell experiment

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  • 3 fertilisations = replicates
    • -
  • two siblings from each fertilisation one control, on FGF treated
    • -
  • sequenced at three time points: S14, S20, S30
    • -
  • 3 x 2 x 3 = 18 groups
    • +

  • Cells were sorted using flow cytometry on the basis of cell surface markers

    • +

  • There are three cell types: LT-HSCs, HSPCs, Progs

    • +

  • Many cells of each cell type were sequenced

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Frog development: background

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🐭 Experimental design

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Schematic of stem cell experiment

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Frog development: Processing so far

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🐭 Aim

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    -
  • selection of cells
    • -
  • selection of genes: subset of surfaceome
    • -
  • log2 normalised values
    • +

  • find genes for cell surface proteins that are important in stem cell identity

    • +

  • Important means genes that are differentially expressed between at least two cell types

    • +

  • Differentially expressed means the expression on one group is significantly higher than the other

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Frog development: Aims

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🐭 Guided analysis

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    -
  • Find interesting cell surface molecule genes that vary between cell types.
    • +

  • The workshops will take you through comparing the HSPC and Prog cells

    • +

  • This is the “least interesting” comparison

    • +

  • You will be guided to carefully document your work so you can apply the same methods to other comparisons

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-

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Deliverables

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  1. Describe the data

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    • Number of cells/samples/reps/treatments
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    • number of genes
      • -
    • type of expression values
      • -
    • prior processing
      • -
    • missing values
      • -
    • overview of expression
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    • clustering of genes/samples
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    2. -

  2. Report on differential expression between two groups

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    • number of DE at 1%, 5% and 10%.
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    • table of expression, fold changes, signifcance at each sig.
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    • Volcano plot
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  3. Report list of marker candidate gene IDs for a cell type of choice. Justify filters. Table with fold FC, p values, IDs, canonical gene names

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  4. Interpret the biology by reporting on a few group of genes and the processes in which they are involved.

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  5. Report on your chosen genes and explain why you think they are good candidates for follow up work

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References

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-Nestorowa, Sonia, Fiona K. Hamey, Blanca Pijuan Sala, Evangelia Diamanti, Mairi Shepherd, Elisa Laurenti, Nicola K. Wilson, David G. Kent, and Berthold Göttgens. 2016. “A Single-Cell Resolution Map of Mouse Hematopoietic Stem and Progenitor Cell Differentiation.” Blood 128 (8): e20–31. https://doi.org/10.1182/blood-2016-05-716480. +
+

🔗 About Omics 1: Hello data!

-
- + + + + + + + + + + + + + + + + + + -
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And GSE81682_HTSeq_counts.txt.zip", - "crumbs": [ - "Week 3: Hello data!", - "Prepare!" - ] + "text": "Frog development: background\n\nRaw data: GEO Series GSE81682\nIllumina HiSeq\nshort reads 150-300bp\nA single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation (Nestorowa et al. 2016)\n3,840 samples\nReads were aligned using G-SNAP and the mapped reads were assigned to Ensembl genes HTSeq\nGSE81682_HTSeq_counts.txt.gz (bottom of the page). 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Metabolomic / Metagenomic data from anaerobic digesters" + }, + { + "objectID": "omics/week-3/study_before_workshop.html#experimental-design", + "href": "omics/week-3/study_before_workshop.html#experimental-design", + "title": "Independent Study to prepare for workshop", + "section": "🐸 Experimental design", + "text": "🐸 Experimental design\n\nSchematic of frog development experiment" + }, + { + "objectID": "omics/week-3/study_before_workshop.html#experimental-design-1", + "href": "omics/week-3/study_before_workshop.html#experimental-design-1", + "title": "Independent Study to prepare for workshop", + "section": "🐸 Experimental design", + "text": "🐸 Experimental design\n\nSchematic of frog development experiment" + }, + { + "objectID": "omics/week-3/study_before_workshop.html#experimental-design-2", + "href": "omics/week-3/study_before_workshop.html#experimental-design-2", + "title": "Independent Study to prepare for workshop", + "section": "🐸 Experimental design", + "text": "🐸 Experimental design\n\nSchematic of frog development experiment\n\n3 fertilisations\ntwo siblings from each fertilisation one control, on FGF treated\nsequenced at three time points\n3 x 2 x 3 = 18 groups" + }, + { + "objectID": "omics/week-3/study_before_workshop.html#section", + "href": "omics/week-3/study_before_workshop.html#section", + "title": "Independent Study to prepare for workshop", + "section": "", + "text": "find important genes\nwhat is ‘important’\n\nexpressed\ndifferentially expressed i.e., which genes are significantly higher in one group vs another\n\n\nthe difference between HSPC and Prog cells the difference between the control and the FGF treated sibling at S30 ## Sequence Data\n\nThe raw data are “reads” from a sequencing machine. Reads are short sequences of DNA or RNA.\nThe reads are aligned to a reference genome or transcriptome. The reads are then counted to quantify the expression of each gene. The counts are normalised to allow comparison between samples.\nreads\nquality control\nalign/pseudoalign\nquantify\nnormalise" + }, + { + "objectID": "omics/week-3/study_before_workshop.html#revise-pivot-longer", + "href": "omics/week-3/study_before_workshop.html#revise-pivot-longer", + "title": "Independent Study to prepare for workshop", + "section": "revise pivot longer", + "text": "revise pivot longer" + }, + { + "objectID": "omics/week-3/study_before_workshop.html#revise-pivot-longer-1", + "href": "omics/week-3/study_before_workshop.html#revise-pivot-longer-1", + "title": "Independent Study to prepare for workshop", + "section": "revise pivot longer", + "text": "revise pivot longer\n\n\n🔗 About Omics 1: Hello data!\n\n\n\nNestorowa, Sonia, Fiona K. Hamey, Blanca Pijuan Sala, Evangelia Diamanti, Mairi Shepherd, Elisa Laurenti, Nicola K. Wilson, David G. Kent, and Berthold Göttgens. 2016. “A Single-Cell Resolution Map of Mouse Hematopoietic Stem and Progenitor Cell Differentiation.” Blood 128 (8): e20–31. https://doi.org/10.1182/blood-2016-05-716480." + }, + { + "objectID": "omics/week-3/study_before_workshop.html#nb-still-in-construction", + "href": "omics/week-3/study_before_workshop.html#nb-still-in-construction", + "title": "Independent Study to prepare for workshop", + "section": "🚧 NB still in construction 🚧", + "text": "🚧 NB still in construction 🚧" + }, + { + "objectID": "omics/week-3/study_before_workshop.html#overview", + "href": "omics/week-3/study_before_workshop.html#overview", + "title": "Independent Study to prepare for workshop", + "section": "Overview", + "text": "Overview\n\n\nConcise summary of the experimental design and aims\nWhat the raw data consist of\nWhat has been done to the data so far\nWhat steps we will take in the workshop" + }, + { + "objectID": "omics/week-3/study_before_workshop.html#experimental-design-3", + "href": "omics/week-3/study_before_workshop.html#experimental-design-3", + "title": "Independent Study to prepare for workshop", + "section": "🐸 Experimental design", + "text": "🐸 Experimental design\n\nSchematic of frog development experiment\n\n3 fertilisations. These are the replicates, .5, .6, A\ntwo siblings from each fertilisation one control, one FGF treated. The treatments are paired\nsequenced at three time points. 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Reads are short sequences of DNA or RNA. +## The Data +There are three datasets -- The reads are aligned to a reference genome or transcriptome. The reads are then counted to quantify the expression of each gene. The counts are normalised to allow comparison between samples. -- reads -- quality control -- align/pseudoalign -- quantify -- normalise +- 🐸 transcriptomic data (bulk RNA-seq) from frog embryos. +- 🐭 transcriptomic data (single cell RNA-seq) from stemcells -## What is a read +- 🍂 ??????? Metabolomic / Metagenomic data from anaerobic digesters -- FASTQ format files - - sequences and information about each sequence's read accuracy +# Experimental design -## Differential expression -- what is differentially expressed -- how are DE genes related -- what are DE genes involved with +## 🐸 Experimental design {auto-animate="true"} -## Stem cells: background +![Schematic of frog development +experiment](images/88H-exp-design-betsy.png){fig-align="center" +width="700"} -Cells were sorted using flow cytometry on the basis of cell surface markers. There are three cell types: +## 🐸 Experimental design {auto-animate="true"} -- long-term haematopoetic stem cells (LT-HSCs) +![Schematic of frog development +experiment](images/88H-exp-design-betsy.png){fig-align="left" +width="200"} -- haematopoetic stem and progenitor cells (HSPCs) +::: incremental +- 3 fertilisations -- progenitor cells (Progs) +- two siblings from each fertilisation one control, on FGF treated -Each cell is then sequenced to quantify all the transcripts in each cell. different transcripts (genes) were identified. +- sequenced at three time points +- 3 x 2 x 3 = 18 groups +::: -## Stem cells: background +## 🐸 Experimental design {auto-animate="true"} -- Raw data: [GEO Series GSE81682](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE81682) -- Illumina HiSeq -- short reads 150-300bp -- [A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5305050/) [@nestorowa2016] -- 3,840 samples -- Reads were aligned using G-SNAP and the mapped reads were assigned to Ensembl genes HTSeq -- GSE81682_HTSeq_counts.txt.gz (bottom of the page). And [GSE81682_HTSeq_counts.txt.zip](../data/jillian/GSE81682_HTSeq_counts.zip) +![Schematic of frog development +experiment](images/88H-exp-design-betsy.png){fig-align="left" +width="200"} -## Stem cells: Processing so far +::: incremental +- 3 fertilisations. [These are the replicates, `.5`, `.6`, + `A`]{style="color:#009900"} -- selection of cells -- selection of genes: subset of surfaceome -- log2 normalised values +- two siblings from each fertilisation one control, one FGF treated. + [The treatments are paired]{style="color:#009900"} -## Stem cells: Aims +- sequenced at three time points. [S14, S20, + S30]{style="color:#009900"} -- Find interesting **cell surface molecule genes** that vary between cell types. +- 3 x 2 x 3 = 18 groups +::: +## 🐸 Aim -## Frog development: background +::: incremental -- 3 fertilisations = replicates -- two siblings from each fertilisation one control, on FGF treated -- sequenced at three time points: S14, S20, S30 -- 3 x 2 x 3 = 18 groups +- find genes important in frog development + +- Important means genes that are differentially expressed between the control and the FGF treated sibling + +- Differentially expressed means the expression on one group is signifcantly higher than the other + +::: + + +## 🐸 Guided analysis + +::: incremental + +- The workshops will take you through comparing the control and FGF treated sibling at S30 + +- This is the "least interesting" comparison + +- You will be guided to carefully document your work so you can apply the same methods to other comparisons + +::: + + + + +## 🐭 Experimental design {auto-animate="true"} + +![Schematic of stem cell experiment](images/88H-exp-design-jillian.png){fig-align="center" +width="700"} + +## 🐭 Experimental design {auto-animate="true"} + +![Schematic of stem cell experiment](images/88H-exp-design-jillian.png){fig-align="left" +width="200"} + +::: incremental +- Cells were sorted using flow cytometry on the basis of cell surface +markers + +- There are three cell types: LT-HSCs, HSPCs, Progs + +- Many cells of each cell type were sequenced + +- +::: + +## 🐭 Experimental design {auto-animate="true"} + +![Schematic of stem cell experiment](images/88H-exp-design-jillian.png){fig-align="left" +width="200"} + +::: incremental + +- There are three cell types: LT-HSCs, HSPCs, Progs [These are the "treaments"]{style="color:#009900"} + + +- Many cells of each cell type were sequenced: [These are the replicates]{style="color:#009900"} + +- [155 LT-HSCs, 701 HSPCs, 798 Progs]{style="color:#009900"} + +::: + +## 🐭 Aim + +::: incremental + +- find genes for cell surface proteins that are important in stem cell identity + +- Important means genes that are differentially expressed between at least two cell types + +- Differentially expressed means the expression on one group is significantly higher than the other + +::: + + +## 🐭 Guided analysis + +::: incremental + +- The workshops will take you through comparing the HSPC and Prog cells + +- This is the "least interesting" comparison + +- You will be guided to carefully document your work so you can apply the same methods to other comparisons + +::: + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -![](images/88H-exp-design-betsy.png) + + + -## Frog development: background + + -- Raw data: [GEO Series GSE81682](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE81682) -- Illumina HiSeq -- short reads 150-300bp -- [A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5305050/) [@nestorowa2016] -- 3,840 samples -- Reads were aligned using G-SNAP and the mapped reads were assigned to Ensembl genes HTSeq -- GSE81682_HTSeq_counts.txt.gz (bottom of the page). And [GSE81682_HTSeq_counts.txt.zip](../data/jillian/GSE81682_HTSeq_counts.zip) + - + + + + -## Frog development: Processing so far -- selection of cells -- selection of genes: subset of surfaceome -- log2 normalised values -## Frog development: Aims -- Find interesting **cell surface molecule genes** that vary between cell types. + + + + + + + + + + + + + + + + + + + + + -## + -## Deliverables + + + + + + + + -1. Describe the data - - Number of cells/samples/reps/treatments - - number of genes - - type of expression values - - prior processing - - missing values - - overview of expression - - clustering of genes/samples + -2. Report on differential expression between two groups - - number of DE at 1%, 5% and 10%. - - table of expression, fold changes, signifcance at each sig. - - Volcano plot + + + -3. Report list of marker candidate gene IDs for a cell type of choice. Justify filters. Table with fold FC, p values, IDs, canonical gene names + + + -4. Interpret the biology by reporting on a few group of genes and the processes in which they are involved. + + -5. Report on your chosen genes and explain why you think they are good candidates for follow up work + + + -## revise pivot longer -## revise pivot longer \ No newline at end of file + diff --git a/update-notes.txt b/update-notes.txt index 0417ddb..e8a7493 100644 --- a/update-notes.txt +++ b/update-notes.txt @@ -46,4 +46,16 @@ for workshop 2, make sure you examine which frog genes are 0 in all of one sampl which genes matters: first do venn diagram, present absent then to differential expressions - +- Raw data: [GEO Series + GSE81682](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE81682) +- Illumina HiSeq +- short reads 150-300bp +- [A single-cell resolution map of mouse hematopoietic stem and + progenitor cell + differentiation](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5305050/) + [@nestorowa2016] +- 3,840 samples +- Reads were aligned using G-SNAP and the mapped reads were assigned + to Ensembl genes HTSeq +- GSE81682_HTSeq_counts.txt.gz (bottom of the page). And + [GSE81682_HTSeq_counts.txt.zip](../data/jillian/GSE81682_HTSeq_counts.zip)