In this example, we investigate a protocol that uses poly-dT primers to reversely transcribe mRNA molecules, that later-on are fragmented by a mechanical shearing known as nebulization. Subsequently, reads are sequenced without PCR or size filtering.
Input
Download
Parameters
| Expression | ||
NB_MOLECULES | 5,000,000 | Number of RNA molecules initially in the experiment |
| TSS_MEAN | 100 | Average deviation from the annotated transcription start site (TSS) |
| POLYA_SCALE | 200 | Scale of the Weibull distribution, shifts the average length of poly-A tail sizes |
| POLYA_SHAPE | 1.5 | 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 | 400 | Minimum length observed after reverse transcription of full-length transcripts |
| RT_MAX | 2,600 | Maximum length observed after reverse transcription of full-length transcripts |
| Fragmentation | ||
| FRAG_SUBSTRATE | DNA | Specifies DNA as the substrate of fragmentation |
| FRAG_METHOD | NB | Nebulization as fragmentation method |
| FRAG_NB_LAMBDA | 600 | Threshold on molecule length that cannot be broken by the shearfield of nebulization |
| FRAG_NB_M | 5 | Strength of the nebulization shearfield (i.e., rotor speed) |
| Amplification and Size Segregation | ||
| PCR_DISTRIBUTION | none | Disable PCR amplification |
| GC_MEAN | NaN | Disable GC bias |
| FILTERING | NO | Disable size filtering |
| Sequencing | ||
| READ_NUMBER | 2,000,000 | Produce 2 million reads |
| READ_LENGTH | 100 | Each read sequence is 100nt long |
| PAIRED_END | NO | Single reads are simulated, one per fragment |
Output
[INFO] I am collecting information on the run.
[INFO] Checking GTF file
*[WARN] Unsorted in line 27 - chr/strand Chr1 + already read.
********* OK (371481:13948:59)
[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/TAIR9_GFF3_genes_sorted.gtf
[GTF FILE] You might want to update your parameters file
[PROFILING] I am assigning the expression profile
********** OK (371481:13948:59)
Reading reference annotation **[WARN] merging exon (-21073927,-21073974) with exon (-21073898,-21073924) in transcript AT1G56280.1 because intervening intron has 4 or less nt.
********[WARN] skipped chromosome ChrM
OK (00:00:03)
found 38564 transcripts
[PROFILING] Parameters
NB_MOLECULES 5000000
EXPRESSION_K -0.6
EXPRESSION_X0 5.0E7
EXPRESSION_X1 9500.0
PRO_FILE_NAME /Users/micha/Desktop/t9_nebulization.pro
profiling ********** OK (00:00:00)
Updating .pro file ********** OK (00:00:00)
molecules 4999389
[LIBRARY] creating the cDNA libary
Initializing Fragmentation File ********** OK (00:00:06)
4999389 mol initialized
[LIBRARY] Reverse Transcription
[LIBRARY] Configuration
Mode: PDT
PWM: No
RT MIN: 400
RT MAX: 2600
Processing Fragments ********** OK (00:00:17)
4999389 mol: in 4999389, new 0, out 4999389
avg Len 1148.562, maxLen 2600
[LIBRARY] Nebulization
[LIBRARY] Configuration
Lambda: 600.0
M: 5.0
Max Length: 2600.0
Recursions: 5
Processing Fragments ********** OK (00:00:32)
8804186 mol: in 4999389, new 3804797, out 8804186
avg Len 652.202, maxLen 2427
start amplification
[LIBRARY] PCR disabled, skipping amplification
Copied results to /Users/micha/Desktop/t9_nebulization.lib
Updating .pro file ********** OK (00:00:00)
[SEQUENCING] getting the reads
Initializing Fragment Index
Indexing ********** OK (00:00:10)
8804186 lines indexed (8804186 fragments, 18951 entries)
sequencing ***[WARN] merging exon (-21073927,-21073974) with exon (-21073898,-21073924) in transcript AT1G56280.1 because intervening intron has 4 or less nt.
*******[WARN] skipped chromosome ChrM
OK (00:10:22)
found 38564 transcripts
[PROFILING] Parameters
NB_MOLECULES 5000000
EXPRESSION_K -0.6
EXPRESSION_X0 5.0E7
EXPRESSION_X1 9500.0
PRO_FILE_NAME /Users/micha/Desktop/t9_nebulization.pro
profiling ********** OK (00:00:00)
Updating .pro file ********** OK (00:00:00)
molecules 4999389
[LIBRARY] creating the cDNA libary
Initializing Fragmentation File ********** OK (00:00:06)
4999389 mol initialized
[LIBRARY] Reverse Transcription
[LIBRARY] Configuration
Mode: PDT
PWM: No
RT MIN: 400
RT MAX: 2600
Processing Fragments ********** OK (00:00:17)
4999389 mol: in 4999389, new 0, out 4999389
avg Len 1148.562, maxLen 2600
[LIBRARY] Nebulization
[LIBRARY] Configuration
Lambda: 600.0
M: 5.0
Max Length: 2600.0
Recursions: 5
Processing Fragments ********** OK (00:00:32)
8804186 mol: in 4999389, new 3804797, out 8804186
avg Len 652.202, maxLen 2427
start amplification
[LIBRARY] PCR disabled, skipping amplification
Copied results to /Users/micha/Desktop/t9_nebulization.lib
Updating .pro file ********** OK (00:00:00)
[SEQUENCING] getting the reads
Initializing Fragment Index
Indexing ********** OK (00:00:10)
8804186 lines indexed (8804186 fragments, 18951 entries)
sequencing ***[WARN] merging exon (-21073927,-21073974) with exon (-21073898,-21073924) in transcript AT1G56280.1 because intervening intron has 4 or less nt.
*******[WARN] skipped chromosome ChrM
OK (00:10:22)
8804186 fragments found (8804186 without PCR duplicates)
3995704 reads sequenced
1251847 reads fall in poly-A tail
3770 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 695 sec.
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