This repository contains the main scripts to calculate the absolute RNA Pol II elongation rates for individual genes based on DRB/TTchem-seq2 data. DRB/TTchem-seq2[1] is an updated version of DRB/TTchem-seq[2], which largely (6-10 folds) increases the number of individual genes whose elongation rates are calculated. The v2 mainly differs from the original version in two aspects:
- The DRB release times have been reduce from 10, 20, 30 and 40 minutes to 5, 10, 15 and 20 minutes.
- In stead of calling the peaks of elongation waves, the wave fronts have been called and represent the distance traveled by RNAPII.
To obtain the elongation rates for singles genes from raw DRB/TTchem-seq2 data, we process the raw sequencing reads using a bash script and then calculated the elongation rates using R scrips.
I. Data processing (bash scripts)
- umi_tools
- samtools
- trimgalore
- cutadapt
- fastqc
- STAR
- subread
- bedtools
- bedGraphToBigWig
- R (DESeq2; rtracklayer; GenomicFeatures; dplyr; tidyr; magrittr; data.table; ggplot2)
- python (pysam)
Download the DRB_TTchem_seq_data_processing.sh script in the script folder and modify the configuration parameters. Then run the script in UNIX system with at least 32G memory.
- UMI attach (optional)
- fastQC & adaptor trimming
- STAR alignment
- Extract uniquely mapping reads
- Deduplication (optional)
- Removal of exon-exon junction mapping reads
- Split forward and reverse reads
- Count mapped reads
- Quantify gene count
- Calculate spike-in size factors
- Convert bam to bigwig
II. Elongation rates calculation (R scripts)
Install the required R packages:
- rtracklayer
- GenomicFeatures
- tibble
- dplyr
- tidyr
- magrittr
- ggplot2
- Download the help_functions.R in the R folder and source it.
- Follow the instructions in elongation_rates.Rmd to calculate gene elongation rates.
- Gregersen LH, Mitter R & Svejstrup JQ (2020) Using TTchem-seq for profiling nascent transcription and measuring transcript elongation. Nat Protoc 15: 604–627