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  • 5.2 Directional RNA-Seq Protocol (H.sapiens)

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Comment: Migrated to Confluence 5.3

Input

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Reference Annotation

Parameter File

Reference Genome

Parameter

Expression
NB_MOLECULES5,000,000Number of RNA molecules initially in the experiment
TSS_MEAN50Average deviation from the annotated transcription start site (TSS)
POLYA_SCALENaNScale of the Weibull distribution, shifts the average length of poly-A tail sizes
POLYA_SHAPENaNShape of the Weibull distribution describing poly-A tail sizes
Fragmentation
FRAG_SUBSTRATERNASpecifies RNA as the substrate of fragmentation
FRAG_METHODURUniform random fragmentation
FRAG_UR_ETA350Average 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
RT_MOTIFdefaultA 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_MEANNaNDisable GC biases
PCR_PROBABILITY0.05PCR duplication probability
FILTERINGNODisable size selection
Sequencing
READ_NUMBER150,000,000Produce 15 million reads
READ_LENGTH75Each read sequence is 75nt long
PAIRED_ENDYESPaired-end reads are simulated (two per fragment)

Output

Code Block
[INFO] I am collecting information on the run.
    initializing profiler  **********
[INFO] Checking GTF file
********** OK (00:00:04)
[PROFILING] I am assigning the expression profile
********** OK (00:00:05)
    Reading reference annotation ********** OK (00:00:08)
    found 34102 transcripts
[PROFILING] Parameters
    NB_MOLECULES    5000000
    EXPRESSION_K    -0.6
    EXPRESSION_X0    5.0E7
    EXPRESSION_X1    9500.0
    PRO_FILE_NAME    /Users/micha/Desktop/hg19_stranded.pro
    profiling ********** OK (00:00:00)
    Updating .pro file  ********** OK (00:00:00)
    molecules    4999517
[LIBRARY] creating the cDNA libary
    Initializing Fragmentation File ********** OK (00:00:07)
    4999517 mol initialized
[LIBRARY] Fragmentation UR
[LIBRARY] Configuration
        D0: 1.0
        Delta:  Not specified, depends on sequence length
        Eta: 350.0
    Processing Fragments ********** OK (00:01:32)
        47836171 mol: in 4999517, new 42836654, out 47836171
        avg Len 315.81723, maxLen 996
    preparing transcript sequences ********** OK (00:01:34)
[INFO] Initializing PWM cache
[INFO] Done
[LIBRARY] Reverse Transcription
[LIBRARY] Configuration
        Mode: RH
        PWM: default
        RT MIN: 500
        RT MAX: 5500
    Processing Fragments ********** OK (00:10:16)
        47890332 mol: in 47836171, new 54161, out 47890332
        avg Len 181.1249, maxLen 1148
        start amplification
[INFO] Loading default PCR distribution
[INFO] Initializing PWM cache
[INFO] Done
[LIBRARY] Amplification
[LIBRARY] Configuration
        Rounds: 15 
        PCR Probability: 0.05 
    Processing Fragments ********** OK (00:02:21)
    Amplification done.
    In: 47890332 Out: 1291507850
        47890332 mol: in 47890332, new 0, out 1291507850
        avg Len 181.17375, maxLen 1127
    Copied results to /Users/micha/Desktop/hg19_stranded.lib
    Updating .pro file  ********** OK (00:00:00)
[SEQUENCING] getting the reads
    Initializing Fragment Index
    Indexing ********** OK (00:00:41)
    25701416 lines indexed (1291507850 fragments, 18682 entries)
    sequencing ********** OK (00:43:11)
    1291507850 fragments found (25701416 without PCR duplicates)
    150003190 reads sequenced
    8101165 reads fall in poly-A tail
    54585326 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 3740 sec.