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 | |
FILTERING | NO | |
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_ENDYES | 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 (371481371580:1394828662:5909) [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 (371481371580:1394828662:5909) 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 49993894999395 [LIBRARY] creating the cDNA libary Initializing Fragmentation File ********** OK (00:00:0604) 4999395 4999389 mol initialized [LIBRARY] Reverse Transcription [LIBRARY] Configuration Mode: PDT PWM: No RT MIN: 400 RT MAX: 2600 Processing Fragments ********** OK (00:00:1718) 4999389 4999395 mol: in 49993894999395, new 0, out 4999389 4999395 avg Len 11481039.5627405, maxLen 2600 [LIBRARY] Nebulization [LIBRARY] Configuration Lambda: 600.0 M: 5.0 Max Length: 2600.0 Recursions: 53 Processing Fragments ********** OK (00:00:3223) 8804186 7498699 mol: in 49993894999395, new 38047972499304, out 8804186 7498699 avg Len 652693.2021967, maxLen 2427 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 (00:00:1009) 88041867498699 lines indexed (88041867498699 fragments, 1895118849 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 (00:1008:2237) 7498699 8804186 fragments found (88041867498699 without PCR duplicates) 39957042001148 reads sequenced 0 1251847 reads fall in poly-A tail 42854 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 695579 sec. |
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