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  • RSS SEQanswers – RNA Sequencing

    • Problem with cummeRbund diffData() May 20, 2013
      Hi all, I'm running Tophat/cufflinks/cuffdiff for differential gene expression and analysis with cummeRbund (v 2.0.0). I'm having an issue with... […]
      Enrique Zudaire
    • How to increase rowsize in heatmap? May 16, 2013
      Hi, I am a complete newbie to all things cummeRbund and am currently fighting with generating readable heatmaps. When I use ... […]
      Mags
    • novoalign mapping May 15, 2013
      Hi, I want to use novoalign to map reads - allowing up to 15 mismatches for 100 bp paired-end reads I am new to novoalign(went through the... […]
      abh
    • Design of expt across multiple lanes May 15, 2013
      Hi, I am performing an RNA-seq experiment to look at differential expression. The design is as follows: 2 populations x 3 biological... […]
      jbono
    • RNA kinds expected in RNA-seq results May 15, 2013
      Hi, We use RNA isolation and library preparation protocols which capture polyadenylated RNA. My question is what kinds of RNA can we expect to... […]
      Kocur
    • Discrepancy between genotype and expressed alleles May 15, 2013
      Hi all, I am working on the analysis of allele-specific expression using both genotype information and RNA-seq data from the same individuals. I... […]
      RedMary

  • RSS Biostar – RNA-Seq

    • How do TopHat options -g , --supress-hits, and Bowtie options interplay?
      Hi, I am currently using TopHat2 to map RNA-seq runs. I think there have been some changes pertaining the -g option. Does anyone know how it works now? I used to think that setting -g would look for n alignments for a given read, report them [if top-scoring] and discard those reads that had more than g [top scoring] alignments. Now, the description sounds mo […]
    • What happened to -k in TopHat for multiple-mapping reads?
      Selecting -g n in tophat does not discard reads mapping more than n, but instead only reports n alignments for those out all all their TOP scoring alignments. I think there used to be an option -k that would allow one to discard reads that topped x alignments -- whatever happened to that? I only see -g in the tophat 2 manual, no reporting options like before […]
    • Does tophat use the library-type information for mapping, or just for the XS flag?
      When I specify library-type to TopHat, i.e., first-strand, second-strand, unstranded, TopHat appends a value + or - to the XS:A tag, which is useful for subsequent analyses, such as annotation. However, does this information influence the "mappability" of reads, or is this unaffected? My guess is that the information will be considered for mapping […]
    • Purpose of Y-shaped adapters in Illumina Sequencing?
      Hi all, Y adapters different sequences to be annealed to the 5' and 3' ends of each molecule in a library. The arms of the Y are unique, and the middle part, connected to the DNA fragment, is complementary. What are the advantages of this? My take of this over having fully-complementary adapters (ADAPTER1 - - - - - ADAPTER1) is that: -Upon primer a […]
    • Cell Type composition in a tissue based on gene marker expression
      I am not sure if the following would even make sense.... Tissues are composed of composite cell types, and often there are studies such as microarray/NGS where we perform a collective sampling of cells from these tissues. Information about the composition (say percentage of cell type) is not taken into consideration. In some case (such as brain/cancer), ther […]
    • Which SNP caller / method to use after aligning RNA-seq with TopHat
      Which SNP caller / method can / should I use after aligning RNA-seq data with TopHat? For genomic data I use GATK, but supposedly it is not just as easy as running GATK on the TopHat RNA-seq data. The team from Broad has no information / documentation on how to use GATK for RNA-seq data. I don't have any variants yet from DNA re-sequencing. […]