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  • 5.1 RNA Hydrolysis Protocol (M.musculus)
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Reference Annotation

Parameter File

Reference Genome


NB_MOLECULES5,000,000Number of RNA molecules initially in the experiment
TSS_MEAN25Average deviation from the annotated transcription start site (TSS)
POLYA_SCALE300Scale of the Weibull distribution, shifts the average length of poly-A tail sizes
POLYA_SHAPE2Shape of the Weibull distribution describing poly-A tail sizes
FRAG_SUBSTRATERNASpecifies RNA as the substrate of fragmentation
FRAG_METHODURUniform random fragmentation

Average expected framgent size after fragmentations, i.e., number of breaks per unit length (exhautiveness of fragmentation)

FRAG_UR_D01Minimum length of fragments produced by UR fragmentation
FRAG_UR_DELTANaNGeometry of molecules in the UR process depends logarithmically on molecule length
Reverse Transcription
RTRANSCRIPTIONYESSwitch on the reverse transcription
RT_PRIMERRHUse random hexamer primers used for first strand synthesis

A default PWM of the current Illumina protocol is used

RT_LOSSLESSYESFlag to force every molecule to be reversely transcribed
RT_MIN500Minimum length observed after reverse transcription of full-length transcripts
RT_MAX5,500Maximum length observed after reverse transcription of full-length transcripts
Amplification and Size Segregation
PCR_DISTRIBUTIONdefaultDefault PCR distribution with 15 rounds and 20 bins
GC_MEAN0.5Mean value of a gaussian distribution that reflects GC bias amplification probability
GC_SD0.1Standard deviation of a gaussian distribution that reflects GC bias amplification probability
FILTERINGYESEnables size filtering of fragments


nullEmploy an empirical Illumina fragment size distribution
SIZE_SAMPLINGMHThe Metropolis-Hastings algorithm is used for filtering
READ_NUMBER15,000,000Produce 15 million reads
READ_LENGTH75Each read sequence is 75nt long
PAIRED_ENDNOSingle reads are simulated (one per fragment)


[INFO] I am collecting information on the run.
    initializing profiler  **********
[INFO] Checking GTF file
********** OK (00:00:03)
[PROFILING] I am assigning the expression profile
********** OK (00:00:05)
    Reading reference annotation ********** OK (00:00:06)
    found 28045 transcripts
[PROFILING] Parameters
    NB_MOLECULES    5000000
    EXPRESSION_K    -0.6
    EXPRESSION_X0    5.0E7
    EXPRESSION_X1    9500.0
    PRO_FILE_NAME    /Users/micha/Desktop/
    profiling ********** OK (00:00:00)
    Updating .pro file  ********** OK (00:00:00)
    molecules    4999480
[LIBRARY] creating the cDNA libary
    Initializing Fragmentation File ********** OK (00:00:06)
    4999480 mol initialized
[LIBRARY] Fragmentation UR
[LIBRARY] Configuration
        D0: 1.0
        Delta:  Not specified, depends on sequence length
        Eta: 170.0
    Processing Fragments ********** OK (00:03:20)
        99433550 mol: in 4999480, new 94434070, out 99433550
        avg Len 154.13617, maxLen 499
    preparing transcript sequences ********** OK (00:02:04)
[INFO] Initializing PWM cache
[INFO] Done
[LIBRARY] Reverse Transcription
[LIBRARY] Configuration
        Mode: RH
        PWM: motif_1mer_0-5.pwm
        RT MIN: 500
        RT MAX: 5500
    Processing Fragments ********** OK (00:18:53)
        99436361 mol: in 99433550, new 2811, out 99436361
        avg Len 226.49129, maxLen 718
        initializing Selected Size distribution
[LIBRARY] Segregating cDNA (Acceptance)
    Processing Fragments ********** OK (00:02:32)
        99436361 mol: in 99436361, new 0, out 3935454
        avg Len 183.18074, maxLen 299
        start amplification
[INFO] Loading default PCR distribution
[INFO] Initializing PWM cache
[INFO] Done
[LIBRARY] Amplification
[LIBRARY] Configuration
        Rounds: 15 
        Mean: 0.5 
        Standard Deviation: 0.1 
    Processing Fragments ********** OK (00:00:19)
    Amplification done.
    In: 3935454 Out: 106111525
        3935454 mol: in 3935454, new 0, out 106111525
        avg Len 183.16734, maxLen 299
    Copied results to /Users/micha/Desktop/mm9_hydrolysis.lib
    Updating .pro file  ********** OK (00:00:00)
[SEQUENCING] getting the reads
    Initializing Fragment Index
    Indexing ********** OK (00:00:03)
    2112053 lines indexed (106111525 fragments, 16421 entries)
    sequencing ********** OK (00:04:11)
    106111525 fragments found (2112053 without PCR duplicates)
    15000653 reads sequenced
    2333333 reads fall in poly-A tail
    511978 truncated reads
    Moving temporary BED file
    Updating .pro file  ********** OK (00:00:00)
    Updating .pro file  ********** OK (00:00:00)
    Updating .pro file  ********** OK (00:00:00)
    Updating .pro file  ********** OK (00:00:00)
[END] I finished, took me 2291 sec.
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