Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • chervil157
    replied
    Oops! I see what you mean now! Feel so embarassed with my lack of understanding!

    Yes just checked and it's all correct - thank you so much!!!!

    Leave a comment:


  • dpryan
    replied
    Err, don't literally type "results()", check it's manual and ensure that it's returning what it's expecting...likely by manually specifying the coefficient (i.e., the "name=something" parameter).

    Leave a comment:


  • chervil157
    replied
    Think I must have done something wrong somehwere because this is what I got:

    > results()
    Error in "results" %in% mcols(mcols(object))$type :
    error in evaluating the argument 'table' in selecting a method for function '%in%': Error in mcols(mcols(object)) :
    error in evaluating the argument 'x' in selecting a method for function 'mcols': Error in mcols(object) :
    error in evaluating the argument 'x' in selecting a method for function 'mcols': Error: argument "object" is missing, with no default

    Leave a comment:


  • dpryan
    replied
    Double check what "results()" is giving you the results for. If it's is indeed giving you the interaction term then you're good to go (you might manually specify the appropriate "name=" coefficient).

    Leave a comment:


  • chervil157
    replied
    Thanks again dpryan! You've been most helpful and really appreciated your comments!

    I went back to read the vignette and now noted the difference between "~Condition + Stimulation" and "~Condition*Stimulation", but when I ran DESeq2 again the result is identical...

    Leave a comment:


  • dpryan
    replied
    I thought you were interested in the interaction between "Condition" and "Stimulation", in which case the design would need to be "~Condition + Stimulation + Condition:Stimulation" (or "~Condition*Stimulation", which is just the short-hand version). Please double check section 3.3 in the vignette.

    Leave a comment:


  • chervil157
    replied
    Followed the vignette and hopefully I have done it correctly:

    > mappedcount <- read.table("CountTable.txt", header=TRUE, row.names=1)
    > coldata <- read.table("Coldata.txt", header=TRUE, row.names=1)
    > Condition = coldata$Infection
    > Stimulation = coldata$Stimulation
    > cds <- DESeqDataSetFromMatrix(countData = mappedcount, colData = coldata, design = ~ Condition + Stimulation)
    > cdsMF <- DESeq(cds)
    > resMF <- results(cdsMF)
    > write.csv(resMF, file="DEG.txt")
    Last edited by chervil157; 02-01-2016, 02:53 AM.

    Leave a comment:


  • chervil157
    replied
    Thank you very much dpryan! I will go through the vignette and see how it goes!

    Last edited by chervil157; 01-31-2016, 03:49 AM.

    Leave a comment:


  • dpryan
    replied
    You don't need any sort of normlization outside of that already provided by DESeq2. Since you're new to things, please have a look through section 1.6 of the DESeq2 vignette. The example they use is essentially identical to yours (in your case, you would use "Infected" and "IFNg" instead of "condition" and "type"). Your design would then be "~Infected*IFNg" and you would be interested in the interaction term, which would tell you the additional effect seen with both the infection and IFNg.

    This method takes care of all of the normalization in a single step and is the statistically preferred method, so your reviewers will be happy

    Edit: See section 3.3 in the vignette too, they explicitly cover interaction terms there. That's exactly what you want.

    Leave a comment:


  • chervil157
    started a topic Normalization to control conditions for DESeq

    Normalization to control conditions for DESeq

    Hi! I am still learning my RNA-Seq analysis and would greatly appreciate some help here.

    Our study is to investigate the pathways associated to interferon-gamma (IFNg)-induced control of infection with bacteriaX. There are four experimental groups (done as 3 biological replicates):

    1) Uninfected without IFNg
    2) Uninfected with IFNg
    3) BacteriaX infected without IFNg
    4) BacteriaX infected with IFNg

    I aligned the data to the hg19 reference genome using TopHat2 followed by HTSeq Count. My question is on the DESeq2 part: what we are really interested is the transcriptional changes associated to both infection and the presence of IFNg.

    Is it possible to normalize condition 3 to condition 1 and condition 4 to condition 2, before performing DESeq2?

    Or are there more approporiate ways to analyze the data?

    Any suggestions will be greatly appreciated! Many thanks in advance!

Latest Articles

Collapse

  • seqadmin
    Essential Discoveries and Tools in Epitranscriptomics
    by seqadmin


    The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist on Modified Bases...
    Today, 07:01 AM
  • seqadmin
    Current Approaches to Protein Sequencing
    by seqadmin


    Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
    04-04-2024, 04:25 PM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, 04-11-2024, 12:08 PM
0 responses
37 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-10-2024, 10:19 PM
0 responses
39 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-10-2024, 09:21 AM
0 responses
35 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-04-2024, 09:00 AM
0 responses
54 views
0 likes
Last Post seqadmin  
Working...
X