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 | 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 |
[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.