Section |
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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. |
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 | |||||
RTRANSCRIPTION | YES | |||||
RT_PRIMER | PDT | |||||
RT_LOSSLESS | YES | |||||
RT_MIN | 400 | |||||
RT_MAX | 2,600 | |||||
PCR_ROUNDS | 13 | |||||
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 | FILTERING | NO|||||
READ_NUMBER | 2,000,000 | Produce 2 million reads | ||||
READ_LENGTH | 100 | PAIRED_END | YESEach read sequence is 100nt long | |||
PAIRED_END | NO | Single reads are simulated, one per fragment |
Code Block |
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[INFO] I am collecting information on the run.
[INFO] Checking GTF file
*[WARN] Unsorted in line 27 - chr/strand Chr1 + already read.
********* OK (371580:28662:09)
[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 (371580:28662:09)
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 4999395
[LIBRARY] creating the cDNA libary
Initializing Fragmentation File ********** OK (00:00:04)
4999395 mol initialized
[LIBRARY] Reverse Transcription
[LIBRARY] Configuration
Mode: PDT
PWM: No
RT MIN: 400
RT MAX: 2600
Processing Fragments ********** OK (00:00:18)
4999395 mol: in 4999395, new 0, out 4999395
avg Len 1039.7405, maxLen 2600
[LIBRARY] Nebulization
[LIBRARY] Configuration
Lambda: 600.0
M: 5.0
Max Length: 2600.0
Recursions: 3
Processing Fragments ********** OK (00:00:23)
7498699 mol: in 4999395, new 2499304, out 7498699
avg Len 693.1967, maxLen 2590
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 (00:00:09)
7498699 lines indexed (7498699 fragments, 18849 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 (00:08:37)
7498699 fragments found (7498699 without PCR duplicates)
2001148 reads sequenced
0 reads fall in poly-A tail
42854 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 579 sec. |