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
Parameter
| Expression | ||
| NB_MOLECULES | 5,000,000 | Number of RNA molecules initially in the experiment |
| TSS_MEAN | 25 | Average deviation from the annotated transcription start site (TSS) |
| POLYA_SCALE | 80 | Scale of the Weibull distribution, shifts the average length of poly-A tail sizes |
| POLYA_SHAPE | 2 | Shape of the Weibull distribution describing poly-A tail sizes |
| Reverse Transcription | ||
| RTRANSCRIPTION | YES | Switch on the reverse transcription |
| RT_PRIMER | PDT | Use poly-dT primers used for first strand synthesis |
| RT_LOSSLESS | YES | Flag to force every molecule to be reversely transcribed |
| RT_MIN | 500 | Minimum length observed after reverse transcription of full-length transcripts |
| RT_MAX | 2,500 | Maximum length observed after reverse transcription of full-length transcripts |
| Fragmentation | ||
| FRAG_SUBSTRATE | DNA | Specifies DNA as the substrate of fragmentation |
| FRAG_METHOD | EZ | Enzymatic digestion as fragmentation method |
| FRAG_EZ_MOTIF | DNAseI.pwm | Fragmentation by enzymatic digestion |
| Amplification and Size Segregation | ||
| PCR_DISTRIBUTION | default | Default PCR distribution with 15 rounds and 20 bins |
| GC_MEAN | 0.5 | Mean value of a gaussian distribution that reflects GC bias amplification probability |
| GC_SD | 0.1 | Standard deviation of a gaussian distribution that reflects GC bias amplification probability |
| FILTERING | YES | Enables size filtering of fragments |
| SIZE_SAMPLING | MH | The Metropolis-Hastings algorithm is used for filtering |
| Sequencing | ||
| READ_NUMBER | 1,000,000 | Produce 1 million reads |
| READ_LENGTH | 36 | Each read sequence is 36nt long |
| PAIRED_END | NO | Single 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.
Overview
Content Tools