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  • questions on FluxSimulator

    hi all, I was playing around with the very useful tool FluxSimulator to generate simulated RNA-Seq reads. And here I have two questions.
    (1) I always got a large fraction of polyA reads (around 35%-38%), which seems too high (pleas see the bottom for the parameters I used). Then I tried to change the parameter "polyA scale" from the default 300 to 30. And I got few polyA reads. So I am a bit confused: 300 should be a reasonable value but why so many polyA reads are generated?

    (2) My second question is about calculation of the ground truth RPKM/FPKM from the simulated reads. Assume M is the total number of reads and n is the number of reads originated from a transcript t, then RPKM=(10^9*n)/(length(t)*M)? The polyA reads should be removed from the calculation. I wonder if this is the way to get the "TRUE" transcript expressions.

    Thank you!
    **********PAR file************************
    REF_FILE_NAME /Users/z/Downloads/FluxSimulator/chr1/another/another.gtf
    PRO_FILE_NAME /Users/z/Downloads/FluxSimulator/chr1/another/another.pro
    LIB_FILE_NAME /Users/z/Downloads/FluxSimulator/chr1/another/another.lib
    SEQ_FILE_NAME /Users/z/Downloads/FluxSimulator/chr1/another/another.bed
    GEN_DIR /Users/zhaohao/Downloads/FluxSimulator/chr1
    TMP_DIR /var/folders/fY/fY+RKWXZHrqzw9LIcH2+3U+++TU/-Tmp-
    NB_MOLECULES 500000
    EXPRESSION_K -0.6
    EXPRESSION_X0 5.0E7
    EXPRESSION_X1 9500.0
    TSS_MEAN 25.0
    POLYA_SHAPE 2.0
    POLYA_SCALE 30.0
    RT_MIN 500
    RT_MAX 5500
    RT_PRIMER RANDOM
    FRAGMENTATION NO
    FRAG_B4_RT NO
    FRAG_MODE PHYSICAL
    FRAG_LAMBDA 900.0
    FRAG_THRESHOLD 0.1
    FILTERING NO
    LOAD_CODING NO
    LOAD_NONCODING YES
    FILT_MIN 200
    FILT_MAX 250
    READ_NUMBER 587916
    READ_LENGTH 75
    PAIRED_END YES

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