TSS assembly pipeline for At_EPDnew_001
This document provides a technical description
of the transcription start site assembly pipeline that was used to
generate EPDnew version 002 for A. thaliana
assembly araTha1 (TAIR10).
Assembly pipeline overview
Description of procedures and intermediate data files
1. Download annotated TSS
Primary annotation data was downloaded from
TAIR the 06-02-2015.
Genes annotations downloaded from TAIR did not contain direct links
to RefSeq ID. For this reason, RefSeq ID has been parsed from
A total number of 31615 promoters were selected.
2. TAIR10 TSS collection
The TAIR10 TSS collection is stored as a tab-deliminated text file
conforming to the SGA format under the name:
The six field contain the following kinds of information:
- NCBI/RefSeq chromosome id
- strand ("+" or "-")
- TAIR ID
Note that the second and forth fields are invariant.
3. Import CAGE data
Data was imported from GEO as BAM file format. BAM files were converted to SGA file format using ChIP-Convert
A step-by-step guide on how to import, map and convert these samples can be found here
5. mRNA 5' tags peak calling
For the only sample present, peak calling for the merged file has been
carried out using ChIP-Peak
on-line tool with the following parameters:
- Window width = 200
- Vicinity range = 200
- Peak refine = Y
- Count cutoff = 9999999
- Threshold = 5
6. TSS validation and shifting
The sample in the collection (mRNA peaks and TAIR10 TSS) was then
processed in a pipeline aiming at validating transcription
start sites with mRNA peaks. A TAIR10 TSS was experimentally confirmed
if an mRNA peak lied in a window of 100 bp around it. The validated
TSS was then shifted to the nearest base with the higher tag
7. TAIR not-validated TSS
The total number (summing up all samples) of non experimentally validated TSS was around 15000.
Transcription Start Sites that mapped closed to other TSS that
belonged to the same gene (100 bp window) were merged into a
unique promoter following the same rule: the promoter that was
validated by the higher number of samples was kept.
9. Final EPDnew collection
The 17000 experimentally validated promoter were stored in the
EPDnew database that can be downloaded from our ftp
site. Scientist are wellcome to use our other tools ChIP-Seq
correlation analysis) and SSA
(for motifs analysis
around promoters) to analyse EPDnew database.