Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Using DeSeq2 with 16s data, valid parameters

    Hello,

    I am trying to use DeSeq2 in R to analyse differential abundance between 16s samples at OTU level. However I am not sure if the code I am using is valid for what I want to do. Codes comes from the phyloseq tutorial. http://joey711.github.io/phyloseq-ex...ns/DESeq2.html

    My data is 16s data (Illumima MiSeq), collected from birds with different caecal lesion scores (0, 1, 2, 3, 4) and uninfected controls, for each I have <10 biological replicates per group. What I am interested in doing in comparing abundance of OTUs across groups.

    I do as below to load my data into the matrix and then to ensure that I only have two groups (eg. 4 & uninfected) for comparison I remove the other groups using the subset option. Then run the code below.

    My question is, is subsetting like this still allowing the program to take all of the data, even that which isn't being compared, into account?

    Secondly am I struggling to know which parameters to use for this type of data with DeSeq2. Can I simply implement what is done for RnaSeq data here to look for differentially abundant OTUs or is this wrong completely? I am not sure which fitType to use; parametric or local or which test to use; Wald or LRT? Doing comparisons there is not much difference in the data, but would be good to know which is best to use.

    The reason I am using DeSeq2 is because I have a small data set, min of 8 biological replicates, max of 11 biological replicates per group.

    Having read the package manual I am still unclear. http://www.bioconductor.org/packages...man/DESeq2.pdf

    library("phyloseq")
    library("ggplot2")
    library("devtools")
    otufile=("table.from_biom_w_consensuslineage_0_replaced.txt")
    mapfile=("Mapping_file_LS.txt")
    trefile=("rep_set_tree.tre")
    qiimedata = import_qiime(otufile, mapfile, trefile)

    qiimedata = subset_samples(qiimedata, LesionScore != "0")
    qiimedata = subset_samples(qiimedata, LesionScore != "1")
    qiimedata = subset_samples(qiimedata, LesionScore != "2")
    qiimedata = subset_samples(qiimedata, LesionScore != "3")

    diagdds = phyloseq_to_deseq2(qiimedata, ~ LesionScore)
    diagdds = DESeq(diagdds, test="Wald", fitType="parametric")

    res = results(diagdds, cooksCutoff = FALSE)
    alpha = 0.05
    sigtab = res[which(res$padj < alpha), ]
    sigtab = cbind(as(sigtab, "data.frame"), as(tax_table(qiimedata)[rownames(sigtab), ], "matrix"))
    head(sigtab)
    sigtabOrdered <- sigtab[order(sigtab$padj),]
    plotMA(res)
    plotDispEsts(diagdds)


    Any help would be great
    Sarah
    Last edited by sarah_27_m; 01-06-2017, 06:54 AM.

  • #2
    The answer to part of my question, this demonstrates that microbiome count data from amplicon sequencing experiments can be treated with the same procedures as for differential expression data from RNA-Seq experiments.

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Strategies for Sequencing Challenging Samples
      by seqadmin


      Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
      03-22-2024, 06:39 AM
    • seqadmin
      Techniques and Challenges in Conservation Genomics
      by seqadmin



      The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

      Avian Conservation
      Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
      03-08-2024, 10:41 AM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, Yesterday, 06:37 PM
    0 responses
    11 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, Yesterday, 06:07 PM
    0 responses
    10 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 03-22-2024, 10:03 AM
    0 responses
    51 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 03-21-2024, 07:32 AM
    0 responses
    68 views
    0 likes
    Last Post seqadmin  
    Working...
    X