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  • 5.3 Poly-dT Priming and DNAse Digestion (S.cerevisiae)
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The simulation of RNA-Seq in Saccharomyces cerevisiae joins a reverse transcription model by poly-dT primers with subsequent fragmenation by DNAseI. Sequence biases that have been reported for the DNAseI fragmentation process (Hansen et al. 2010) are captured in the simulation by a position weight matrix (DNAseI.pwm).

Input

Download


Reference Annotation

Parameter File

Reference Genome

Parameter

Expression
NB_MOLECULES5,000,000Number of RNA molecules initially in the experiment
TSS_MEAN25Average deviation from the annotated transcription start site (TSS)
POLYA_SCALE80Scale of the Weibull distribution, shifts the average length of poly-A tail sizes
POLYA_SHAPE2Shape of the Weibull distribution describing poly-A tail sizes
Reverse Transcription
RTRANSCRIPTIONYESSwitch on the reverse transcription
RT_PRIMERPDTUse poly-dT primers used for first strand synthesis
RT_LOSSLESSYESFlag to force every molecule to be reversely transcribed
RT_MIN500

Minimum length observed after reverse transcription of full-length transcripts

RT_MAX2,500Maximum length observed after reverse transcription of full-length transcripts
Fragmentation
FRAG_SUBSTRATEDNASpecifies DNA as the substrate of fragmentation
FRAG_METHODEZEnzymatic digestion as fragmentation method
FRAG_EZ_MOTIFDNAseI.pwmFragmentation by enzymatic digestion
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
SIZE_SAMPLINGMHThe Metropolis-Hastings algorithm is used for filtering
Sequencing
READ_NUMBER1,000,000Produce 1 million reads
READ_LENGTH36Each read sequence is 36nt long
PAIRED_ENDNOSingle reads are simulated, one per fragment

Output

[INFO] I am collecting information on the run.
    initializing profiler  **********
[INFO] Checking GTF file
*[WARN] Unsorted in line 5 - cannot perform gene clustering: chrI + YAL069W @ 335 after YAL012W @ 130799
********* OK (00:00:02)
[GTF FILE] The GTF reference file given is not sorted, but we found a sorted version.
[GTF FILE] The Simulator will use /Users/micha/Desktop/sacCer3_SGDGenes_fromUCSC120515_sorted.gtf
[GTF FILE] You might want to update your parameters file
[PROFILING] I am assigning the expression profile
********** OK (00:00:02)
    Reading reference annotation *[WARN] merging exon (31229,35248) with exon (29935,31227) in transcript YBL100W-B because intervening intron has 4 or less nt.
[WARN] merging exon (222636,226598) with exon (221330,222634) in transcript YBL005W-B because intervening intron has 4 or less nt.
*********[WARN] merging exon (-854953,-856257) with exon (-850989,-854951) in transcript YPR158C-D because intervening intron has 4 or less nt.
 OK (00:00:01)
    found 6664 transcripts
[PROFILING] Parameters
    NB_MOLECULES    5000000
    EXPRESSION_K    -0.6
    EXPRESSION_X0    5.0E7
    EXPRESSION_X1    9500.0
    PRO_FILE_NAME    /Users/micha/Desktop/sacCer3_enzyme.pro
    profiling ********** OK (00:00:00)
    Updating .pro file  ********** OK (00:00:00)
    molecules    4999971
[LIBRARY] creating the cDNA libary
    Initializing Fragmentation File ********** OK (00:00:04)
    4999971 mol initialized
[LIBRARY] Reverse Transcription
[LIBRARY] Configuration
        Mode: PDT
        PWM: No
        RT MIN: 500
        RT MAX: 2500
    Processing Fragments ********** OK (00:00:15)
        4999971 mol: in 4999971, new 0, out 4999971
        avg Len 969.7831, maxLen 2500
    preparing transcript sequences *[WARN] merging exon (31229,35248) with exon (29935,31227) in transcript YBL100W-B because intervening intron has 4 or less nt.
*********[WARN] merging exon (-854953,-856257) with exon (-850989,-854951) in transcript YPR158C-D because intervening intron has 4 or less nt.
 OK (00:00:02)
[LIBRARY] Enzymatic Digestion
[LIBRARY] Configuration
Left Flank : 100
Right Flank : 300
Motif: DNAseI.pwm
    Processing Fragments ********** OK (00:02:38)
        60604099 mol: in 4999971, new 55604128, out 60604099
        avg Len 80.00923, maxLen 2500
        initializing Selected Size distribution
[LIBRARY] Segregating cDNA (MCMC Filter)
    Processing Fragments ********** OK (00:01:47)
        60604099 mol: in 60604099, new 0, out 25719279
        avg Len 47.310493, maxLen 276
        start amplification
[INFO] Loading default PCR distribution
[LIBRARY] Amplification
[LIBRARY] Configuration
        Rounds: 15 
        Mean: 0.5 
        Standard Deviation: 0.1 
    Processing Fragments ********** OK (00:01:05)
    Amplification done.
    In: 25719279 Out: 693695450
        25719279 mol: in 25719279, new 0, out 693695450
        avg Len 47.319595, maxLen 266
    Copied results to /Users/micha/Desktop/sacCer3_enzyme.lib
    Updating .pro file  ********** OK (00:00:00)
[SEQUENCING] getting the reads
    Initializing Fragment Index
    Indexing ********** OK (00:00:14)
    13804020 lines indexed (693695450 fragments, 6534 entries)
    sequencing *[WARN] merging exon (31229,35248) with exon (29935,31227) in transcript YBL100W-B because intervening intron has 4 or less nt.
*********[WARN] merging exon (-854953,-856257) with exon (-850989,-854951) in transcript YPR158C-D because intervening intron has 4 or less nt.
 OK (00:14:03)
    693695450 fragments found (13804020 without PCR duplicates)
    998612 reads sequenced
    226528 reads fall in poly-A tail
    407504 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 1305 sec.
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