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  • Problems with RNA-seq analysis results

    Hi everyone,

    This is my first post here, be sure to let me know if I break a rule of conduct or anything. Tricks & Tips are appreciated.

    The situation:
    I'm currently trying to analyze RNA-seq data from Illumina Body Map 2.0. I've built a pipeline that seems reasonable and used it to asses quality, trim, map, analyze RNA-seq data with the standard tools. The pipeline is for 100bp single end reads.

    The pipeline:
    Quality (fastx_tools for trimming and filtering, FastQC for reporting)
    fastq_quality_filter -Q33 -q 20 -p 80 <FASTQC_FILE>
    fastq_quality_trimmer -Q33 -t 20 -l 50 <FILTER_OUT>
    fastqc <TRIM_OUT>

    Assembling
    tophat --solexa-quals <UCSC hg19 REF> <TRIM_OUT>

    Analysis
    cufflinks <TOPHAT_OUT>
    cuffcompare -r <UCSC hg19 ANNOTATION> -R <CUFFLINKS_OUT>

    The problems
    The output of cuffcompare (cuffcmp.tacking) identifies:
    13586 [23.32%] novel (class code j)
    6127 [10.51%] intronic (class code i)
    19145 [32.86%] contained (class code c)

    In this sample, novel+intronic > contained. I'm highly dubious of the trustfulness of those results since one would not expect such high number of non previously reported transcripts. If anyone could point out a flaw in the pipeline or my interpretation of the obtained results I would greatly appreciate it. Do tell if I need to give more details on any part.

    Best regards,

    Simon
    Last edited by spapillon; 11-29-2011, 09:48 PM.

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