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  • Tophat2 Bowtie2 Htseq-count for bacteria

    Hey this is my first try to analyse a rna-seq project. Since the company we worked with is not able to give me a usefull annotated differential expression table...

    I just want to know for sure, if its half-way right what I do.

    My samples: 2 conditions, 2 replicats from each condition, 50bp single-end, not strand-specific. I got 23m-50m reads per library.

    I want to know differential expressed genes between conditions.

    I first wanted to use bowtie2 for alignment and that worked pretty well until I noticed that no NH tag for htseqcount is written.

    So I switched to tophat and there it got complicated:

    In default tophat2 finds lesser alignments than bowtie2. Why? As I understand tophat2 uses bowtie2 for aligment.

    As I dont want to find novel junctions, as there is no splicing my bacterium, the final command I used after several attempts is:

    tophat2 -G file.gtf --no-novel-juncs --no-coverage-search --library-typ fr-unstranded index file.fastq

    With every attempt (first: --no-coverage-search; second: added -G; third: added --no-novel juncs) the count of aligned reads dropped a little bit. Why it droppend between this 3 modes?

    With the last mode I got an alignment rate of 65-75%.

    I finally got my count tables with
    samtools view file.bam | htseq-count -t gene -s no - file.gtf > counts.txt

    Now I will use deseq for differential expression.

    Everything ok so far?

  • #2
    I just went through the same process as what you did
    see my post here http://crazyhottommy.blogspot.com/20...-sort-and.html

    I am following the protocol from Simon Anders http://www.nature.com/nprot/journal/....2013.099.html
    Count-based differential expression analysis of RNA sequencing data using R and Bioconductor






    Originally posted by chickenmcfu View Post
    Hey this is my first try to analyse a rna-seq project. Since the company we worked with is not able to give me a usefull annotated differential expression table...

    I just want to know for sure, if its half-way right what I do.

    My samples: 2 conditions, 2 replicats from each condition, 50bp single-end, not strand-specific. I got 23m-50m reads per library.

    I want to know differential expressed genes between conditions.

    I first wanted to use bowtie2 for alignment and that worked pretty well until I noticed that no NH tag for htseqcount is written.

    So I switched to tophat and there it got complicated:

    In default tophat2 finds lesser alignments than bowtie2. Why? As I understand tophat2 uses bowtie2 for aligment.

    As I dont want to find novel junctions, as there is no splicing my bacterium, the final command I used after several attempts is:

    tophat2 -G file.gtf --no-novel-juncs --no-coverage-search --library-typ fr-unstranded index file.fastq

    With every attempt (first: --no-coverage-search; second: added -G; third: added --no-novel juncs) the count of aligned reads dropped a little bit. Why it droppend between this 3 modes?

    With the last mode I got an alignment rate of 65-75%.

    I finally got my count tables with
    samtools view file.bam | htseq-count -t gene -s no - file.gtf > counts.txt

    Now I will use deseq for differential expression.

    Everything ok so far?

    Comment


    • #3
      Yes, the description of Deseq is easy to follow, even with no R experience.

      Unfortunately I have run in other problems. Deseq analysis gives me 125 differentially regulated genes. The commercial sequencing service gives me a cuffdiff result of 200 genes (mixed up annotation though). So now I went through the process of cuffdiff and got 129 regulated genes.

      As I now have conducted nearly every possible mapping (bowtie2, tophat2), whole genome or with options for tophat -G and -T and then for cuffdiff -M with the rtRNA.gtf, I could bring it up to 136.

      The only thing in which my analysis differs from them is, that they align straight to only the cds and ncRNAs, completely disregarding the RNAs, but then give cuffdiff a full gtf. They afterwards did a second mapping with the fastqs to rtRNAs, so I know that up to 4.5% map to them.

      Is this right? Can this difference within the alignment give such a huge difference in the results?

      Comment

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