Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • gareth.lim
    replied
    Sorry clicked too fast...

    Sorry I should add that so far I have done this:
    >WT<-"C1"
    > mut<-c("C2","C3","C4","C5","C6")

    Originally posted by gareth.lim View Post
    My experiment is complicated (at least in my mind =) ). I have three time points (0,24,48 hrs) and two conditions (siControl and siTarget) for a total of 6 groups. I'm trying to see how knockdown of my target affects the transcriptome at each time point.

    This information is the closest I can find online that describes how to make a log2-fold change heat map and a dendrogram.... but I'm having some troubles. Can anyone please help?

    A few questions so far:

    1. sigGenes <- getSig(cuff,'BY4741','ino80',alpha=0.05,level='genes')
    -Why were only two of your groups selected here and not the whole data set? Does this impact the final result?

    2. db <- logFC(db,mutants=mut,WT=WT,logBase=2,pseudo=1)
    -when i enter this, I get an error message: Error in db[, mutants] : incorrect number of dimensions

    Leave a comment:


  • gareth.lim
    replied
    My experiment is complicated (at least in my mind =) ). I have three time points (0,24,48 hrs) and two conditions (siControl and siTarget) for a total of 6 groups. I'm trying to see how knockdown of my target affects the transcriptome at each time point.

    This information is the closest I can find online that describes how to make a log2-fold change heat map and a dendrogram.... but I'm having some troubles. Can anyone please help?

    A few questions so far:

    1. sigGenes <- getSig(cuff,'BY4741','ino80',alpha=0.05,level='genes')
    -Why were only two of your groups selected here and not the whole data set? Does this impact the final result?

    2. db <- logFC(db,mutants=mut,WT=WT,logBase=2,pseudo=1)
    -when i enter this, I get an error message: Error in db[, mutants] : incorrect number of dimensions

    Leave a comment:


  • hubery_Bio
    replied
    maybe the program cluster can help u. you just need to change the format of the cuffdiff result.

    Leave a comment:


  • jp.
    replied
    Hi
    Would you post your commands to make dendrogram in csHeatmap ?
    I just can not add dendrogram using csHeatmap.
    You used heatmap.2(.... can read cuffdiff out and make heatmap with dendrogram ?
    Thank you

    Originally posted by devking View Post
    Hi Tir,

    Thanks for your response! I have been using cummeRbund and it has been very easy to use so far to make heatmaps of gene expression. But I didn't immediately see a way to look at *fold-change* expression between two conditions. (I've never used something like R before so things that should be simple usually aren't for me ). However, on the advice of one of the cummeRbund authors, Loyal, I was able to make log fold-change heat maps by extracting raw FPKM values from fpkmMatrix(), do the log2FC transformation in R, then use heatmap.2() from gplots to create something like the attached file.

    Leave a comment:


  • jp.
    replied
    Hi
    Can you please give me your commands to make csHeatmap using cummeRbund ?
    I tried making the using cummeRbund and succeeded, however, can not add dandogram and change color which you have done it.
    May you please write back to me in little detail because I am a beginer.
    Thank you in advance.
    Jp.


    Originally posted by devking View Post
    Hi Tir,

    Thanks for your response! I have been using cummeRbund and it has been very easy to use so far to make heatmaps of gene expression. But I didn't immediately see a way to look at *fold-change* expression between two conditions. (I've never used something like R before so things that should be simple usually aren't for me ). However, on the advice of one of the cummeRbund authors, Loyal, I was able to make log fold-change heat maps by extracting raw FPKM values from fpkmMatrix(), do the log2FC transformation in R, then use heatmap.2() from gplots to create something like the attached file.

    Leave a comment:


  • emolinari
    replied
    Thanks Devking!
    This really helps me out!!!

    Leave a comment:


  • devking
    replied
    Maybe this can help you get started:


    Code:
    library(cummeRbund); library(gplots)
    cuff <- readCufflinks("cuffdiff_out",rebuild=T)
    db <- fpkmMatrix(genes(cuff))
    sigGenes <- getSig(cuff,'BY4741','ino80',alpha=0.05,level='genes')
    db <- db[sigGenes,]	
    
    
    WT <- "BY4741" # Set the column name of the WT sample
    mut <- c("ino80","arp5","ies6") # Set column names of test conditions you want in the analysis
    
    logFC <- function(db,mutants,WT,logBase=2,pseudo=1) {
    		if (length(WT) !=1 ) {
    			stop('WT must refer to a single gene/column')
    			}
    		if (is.numeric(logBase)==FALSE) {
    			stop('logBase must be a numeric value')
    			}
    		
    		db <- (db[,mutants]+pseudo)/(db[,WT]+pseudo)
    		db <- log(db,logBase)
    		
    }
    
    db <- logFC(db,mutants=mut,WT=WT,logBase=2,pseudo=1) # This does the log transformation
    db <- as.matrix(db)
    
    heatmap.2(
    		db,
    		Rowv=TRUE,
    		Colv=FALSE,
    		dendrogram="row",
    		trace="none",
    		labRow="",
    		density.info=c("none"),
    		main="Log(Fold-Change) \nExpression Profiles"
    	)

    Leave a comment:


  • emolinari
    replied
    Originally posted by devking View Post
    I quantified expression using the 'Tuxedo' protocol (http://www.nature.com/nprot/journal/....2012.016.html) and used a custom R script to generate the heatmap. Using cummeRbund, you can get a matrix of FPKM expression values, and then use heatmap.2() in the R 'gplots' package for the heatmap call. You could also import your expression matrix into Java TreeView which also plots nice heatmaps. Hope this helps!
    Thanks for the answer...I actually have followed the same path, but I have just been able to produce a differential expression heatmap.

    I'll see what I can do for up and down regulation...unfortunately java is too hardcore informatics for me!!!
    Thanks
    Manu

    Leave a comment:


  • devking
    replied
    I quantified expression using the 'Tuxedo' protocol (http://www.nature.com/nprot/journal/....2012.016.html) and used a custom R script to generate the heatmap. Using cummeRbund, you can get a matrix of FPKM expression values, and then use heatmap.2() in the R 'gplots' package for the heatmap call. You could also import your expression matrix into Java TreeView which also plots nice heatmaps. Hope this helps!

    Leave a comment:


  • emolinari
    replied
    Originally posted by devking View Post
    Hi Tir,

    Thanks for your response! I have been using cummeRbund and it has been very easy to use so far to make heatmaps of gene expression. But I didn't immediately see a way to look at *fold-change* expression between two conditions. (I've never used something like R before so things that should be simple usually aren't for me ). However, on the advice of one of the cummeRbund authors, Loyal, I was able to make log fold-change heat maps by extracting raw FPKM values from fpkmMatrix(), do the log2FC transformation in R, then use heatmap.2() from gplots to create something like the attached file.
    Hi Devking,

    I was looking to do the same plot as you did...is it done with DESeq?
    did you use a customized R script?
    Thanks!!!
    Manu

    Leave a comment:


  • devking
    replied
    Originally posted by NicoBxl View Post
    Did you try DESeq ? There is an interesting clustering section in the DESeq vignette on bioconductor.
    Hi Nico,

    Thanks for the tip. It seems like all of the differential gene expression statistical methods (e.g. cuffdiff, DESeq, edgeR, NOIseq etc) return slightly different sets of significantly differentially expressed genes. E.g. check out:


    So I was actually planning on using DESeq next and comparing the results with my cuffdiff data. I was thinking of just focusing on the genes that tested significant for DE from both methods. Not sure if this is reasonable or worth the time...

    Best,
    Devin

    Leave a comment:


  • devking
    replied
    Originally posted by tir_al View Post
    As you are already using the tuxedo pipeline, you could take a look at the cummeRbund
    Hi Tir,

    Thanks for your response! I have been using cummeRbund and it has been very easy to use so far to make heatmaps of gene expression. But I didn't immediately see a way to look at *fold-change* expression between two conditions. (I've never used something like R before so things that should be simple usually aren't for me ). However, on the advice of one of the cummeRbund authors, Loyal, I was able to make log fold-change heat maps by extracting raw FPKM values from fpkmMatrix(), do the log2FC transformation in R, then use heatmap.2() from gplots to create something like the attached file.
    Attached Files

    Leave a comment:


  • devking
    replied
    Originally posted by jordiet View Post
    Hi devking,

    sorry I can't help you with your CuffDiff issue. Have you used BY4741 as a reference genome for your alignments? I am working with the same strain and I would like to use it as a reference genome to align my evolved strains to this one. Do you know where can I find an assembled genome of BY4741 to use as a reference?
    Hi Jordiet

    For my analysis I was only interested in quantifying expression according to a known and annotated genome so I used the Ensembl release 69 EF4 genome.

    I figured since the transcripts in yeast are so well-annotated already it seemed like more trouble than it was worth to assemble a new transcriptome using cufflinks/cuffmerge.

    I might be misunderstanding your question, but it seems like you're more interested in doing a full analysis as outlined in the nat protocol paper "Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks" in which you'll have your own assembled transcriptome annotation to quantify.

    check the paper: http://www.nature.com/nprot/journal/....2012.016.html

    Best,
    Devin

    Leave a comment:


  • NicoBxl
    replied
    Did you try DESeq ? There is an interesting clustering section in the DESeq vignette on bioconductor.

    It's pretty simple to use :

    1. align
    2. Extract read count with htseq-count
    3. Use DESeq to perform DE analysis and plot some results

    Check : http://bioconductor.org/packages/rel...tml/DESeq.html

    Leave a comment:


  • tir_al
    replied
    Dear Devkin,

    The heatmap you posted was done in R using the ggplot2 package.

    As you are already using the tuxedo pipeline, you could take a look at the cummeRbund

    which should enable you to get easy data integration.

    Best

    Leave a comment:

Latest Articles

Collapse

  • seqadmin
    Exploring the Dynamics of the Tumor Microenvironment
    by seqadmin




    The complexity of cancer is clearly demonstrated in the diverse ecosystem of the tumor microenvironment (TME). The TME is made up of numerous cell types and its development begins with the changes that happen during oncogenesis. “Genomic mutations, copy number changes, epigenetic alterations, and alternative gene expression occur to varying degrees within the affected tumor cells,” explained Andrea O’Hara, Ph.D., Strategic Technical Specialist at Azenta. “As...
    07-08-2024, 03:19 PM
  • seqadmin
    Exploring Human Diversity Through Large-Scale Omics
    by seqadmin


    In 2003, researchers from the Human Genome Project (HGP) announced the most comprehensive genome to date1. Although the genome wasn’t fully completed until nearly 20 years later2, numerous large-scale projects, such as the International HapMap Project and 1000 Genomes Project, continued the HGP's work, capturing extensive variation and genomic diversity within humans. Recently, newer initiatives have significantly increased in scale and expanded beyond genomics, offering a more detailed...
    06-25-2024, 06:43 AM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, 07-19-2024, 07:20 AM
0 responses
40 views
0 likes
Last Post seqadmin  
Started by seqadmin, 07-16-2024, 05:49 AM
0 responses
52 views
0 likes
Last Post seqadmin  
Started by seqadmin, 07-15-2024, 06:53 AM
0 responses
64 views
0 likes
Last Post seqadmin  
Started by seqadmin, 07-10-2024, 07:30 AM
0 responses
43 views
0 likes
Last Post seqadmin  
Working...
X