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.
NB_MOLECULES | 5,000,000 |
TSS_MEAN | 100 |
POLYA_SCALE | 200 |
POLYA_SHAPE | 1.5 |
FRAG_SUBSTRATE | DNA |
FRAG_METHOD | NB |
FRAG_NB_LAMBDA | 600 |
FRAG_NB_M | 5 |
RTRANSCRIPTION | YES |
RT_PRIMER | PDT |
RT_LOSSLESS | YES |
RT_MIN | 400 |
RT_MAX | 2,600 |
FILTERING | NO |
READ_NUMBER | 2,000,000 |
READ_LENGTH | 100 |
PAIRED_END | YES |
[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.